Patched genes and uses related thereto

ABSTRACT

Methods for isolating patched genes, including the mouse and human patched genes, as well as invertebrate patched genes and sequences, are provided. Decreased expression of patched is associated with the occurrence of human cancers, particularly basal cell carcinomas of the skin. The patched and hedgehog genes are useful in creating transgenic animal models for these human cancers. The patched nucleic acid compositions find use in identifying homologous or related proteins and the DNA sequences encoding such proteins; in producing compositions that modulate the expression or function of the protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as treatment of cancer, identification of cell type based on expression, and the like. The DNA is further used as a diagnostic for a genetic predisposition to cancer, and to identify specific cancers having mutations in this gene.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/724,631, filed on Nov. 28, 2000, now U.S. Pat. No. 6,551,782, whichis a continuation of Ser. No. 08/918,658, filed on Aug. 22, 1997, nowU.S. Pat. No. 6,429,354, which is a continuation-in-part of U.S. Ser.No. 08/656,055, filed on May 31, 1996, now U.S. Pat. No. 6,027,882 whichis a continuation-in-part of U.S. Ser. No. 08/540,406, filed on Oct. 6,1995, now U.S. Pat. No. 5,837,538, which is a continuation-in-part ofU.S. application Ser. No. 08/319,745, filed on Oct. 7, 1994, (nowabandoned). The specifications of each of these prior applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Segment polarity genes were originally discovered as mutations in fliesthat change the pattern of body segment structures. Mutations in thesegenes cause animals to develop changed patterns on the surfaces of bodysegments; the changes affecting the pattern along the head to tail axis.Among the genes in this class are hedgehog, which encodes a secretedprotein (HH), and patched, which encodes a protein structurally similarto transporter proteins, having twelve transmembrane domains (ptc), withtwo conserved glycosylation signals.

The hedgehog gene of flies has at least three vertebrate relatives—Sonichedgehog (Shh); Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Shh isexpressed in a group of cells, at the posterior of each developing limbbud, that have an important role in signaling polarity to the developinglimb. The Shh protein product, SHH, is a critical trigger of posteriorlimb development, and is also involved in polarizing the neural tube andsomites along the dorsal ventral axis. Based on genetic experiments inflies, patched and hedgehog have antagonistic effects in development.The patched gene product, ptc, is widely expressed in fetal and adulttissues, and plays an important role in regulation of development. Ptcdownregulates transcription of itself, members of the transforminggrowth factor and Wnt gene families, and possibly other genes. Amongother activities, HH upregulates expression of patched and other genesthat are negatively regulated by patched.

It is of interest that many genes involved in the regulation of growthand control of cellular signaling are also involved in oncogenesis. Suchgenes may be oncogenes, which are typically upregulated in tumor cells,or tumor suppressor genes, which are down-regulated or absent in tumorcells. Malignancies may arise when a tumor suppressor is lost and/or anoncogene is inappropriately activated. Familial predisposition to cancermay occur when there is a mutation, such as loss of an allele encoding asuppressor gene, present in the germline DNA of an individual.

The most common form of cancer in the United States is basal cellcarcinoma of the skin. While sporadic cases are very common, there arealso familial syndromes, such as the basal cell nevus syndrome (BCNS).The familial syndrome has many features indicative of abnormal embryonicdevelopment, indicating that the mutated gene also plays an importantrole in development of the embryo. A loss of heterozygosity ofchromosome 9q alleles in both familial and sporadic carcinomas suggeststhat a tumor suppressor gene is present in this region. The highincidence of skin cancer makes the identification of this putative tumorsuppressor gene of great interest for diagnosis, therapy, and drugscreening.

Relevant Literature

Descriptions of patched, by itself or its role with hedgehog may befound in Hooper and Scott (1989) Cell 59-.751-765; and Nakano et al.(1989) Nature 341-.508-513. Both of these references also describe thesequence for Drosophila patched. Discussions of the role of hedgehoginclude Riddle et al. (1993) Cell 75-.1401-1416-, Echelard et al. (1993)Cell 75:1417-1430-Krauss et al. (1993) Cell 75:1431-1444 (1993); Tabataand Kornberg (1994) 76:89-102; Heemskerk and DiNardo (1994) Cell76:449-460; and Roelink et al. (1994) Cell 76:-761-775.

Mapping of deleted regions on chromosome 9 in skin cancers is describedin Habuchi et al. (1995) Oncogene 11: 1671-1674, Quinn et al. (1994)Genes Chromosome Cancer 11:222-225; Quinn et al. (1994) J. Invest.Dermatol. 102:300-303; and Wicking et al. (1994) Genomics 22:505-511.

Gorlin (1987) Medicine 66:98-113 reviews nevoid basal cell carcinomasyndrome. The syndrome shows autosomal dominant inheritance withprobably complete penetrance. About 60% of the cases represent newmutations. Developmental abnormalities found with this syndrome includerib and craniofacial abnormalities, polydactyly, syndactyly and spinabifida. Tumors found with the syndrome include basal cell carcinomas,fibromas of the ovaries and heart, cysts of the skin, jaws andmesentery, meningiomas and medulloblastomas.

SUMMARY OF THE INVENTION

Isolated nucleotide compositions and sequences are provided for patched(ptc) genes, including mammalian, e.g. human and mouse, and invertebratehomologs. Decreased expression of ptc is associated with the occurrenceof human cancers, particularly basal cell carcinomas and other tumors ofepithelial tissues such as the skin. The cancers may be familial, havingas a component of risk a germline mutation in the gene, or may besporadic. Ptc, and its antagonist hedgehog, are useful in creatingtransgenic animal models for these human cancers. The ptc nucleic acidcompositions find use in identifying homologous or related genes; inproducing compositions that modulate the expression or function of itsencoded protein, ptc; for gene therapy; mapping functional regions ofthe protein- and in studying associated physiological pathways. Inaddition, modulation of the gene activity in vivo is used forprophylactic and therapeutic purposes, such as treatment of cancer,identification of cell type based on expression, and the like. Ptc,anti-ptc antibodies and ptc nucleic acid sequences are useful asdiagnostics for a genetic predisposition to cancer or developmentalabnormality syndromes, and to identify specific cancers having mutationsin this gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph having a restriction map of about 10 kbp of the 5′region upstream from the initiation codon of Drosophila patched gene andbar graphs of constructs of truncated portions of the 5′ region joinedto fl-galactosidase, where the constructs are introduced into fly celllines for the production of embryos. The expression of fl-gal in theembryos is indicated in the right-hand table during early and latedevelopment of the embryo. The greater the number of +'s, the moreintense the staining.

FIG. 2 shows a summary of mutations found in the human patched genelocus that are associated with basal cell nevus syndrome. Mutation (1)is found in sporadic basal cell carcinoma, and is a C to T transition inexon 3 at nucleotide 523 of the coding sequence, changing Leu 175 to Phein the first extracellular loop. Mutations 2-4 are found in hereditarybasal carcinoma nevus syndrome. (2) is an insertion of 9 bp atnucleotide 2445, resulting in the insertion of an additional 3 aminoacids after amino acid 815. (3) is a deletion of 11 bp, which removes nt2442-2452 from the coding sequence. The resulting frameshift truncatesthe open reading frame after amino acid 813, just after the seventhtransmembrane domain. (4) is a G to C alteration that changes twoconserved nucleotides of the 3′ splice site adjacent to exon 10,creating a non-functional splice site that truncates the protein afteramino acid 449, in the second transmembrane region.

FIG. 3 (panels A-B) illustrates the generation of ptc mutations. (A) Theptc mutant allele was generated by homologous recombination between theKO1 targeting vector and ptc. External probe A detected a 3′ EcoRVpolymorphism on blots and probe B detected a 5′ SacI polymorphism. Exonsare numbered. (B) Transmission of the ptc^(KO1) allele through thegermline was confirmed by Southern blot (upper panel) and a PCRgenotyping assay (lower panel). PCR primers are indicated as arrows inA. Because the homozygous mutant embryos were being resorbed, there wasmuch less yolk sac DNA in the −/− lanes.

FIG. 4 (panels A-G) illustrate the germ layer-specific derepression ofHh target genes in ptc^(−/−) embryos. (A, B) Lateral views of E8.25wild-type (A) and ptc^(−/−) (B) embryos. The headfolds are overgrown inthe mutant (white arrows) and the heart is not properly formed (redarrows). (C) Lateral views of E8.75 ptc^(+/−) (left) and ptc^(−/−)(right) embryos stained with X-gal (28) (D, E, F, G) Transverse sectionsthrough E8.75 ptc^(+/−) (D, F) and ptc^(−/−) (E, G) embryos stained withX-gal (D, E) or hybridized with a digoxigenin labeled Gli probe (29) (F,G). Both lacZ and Gli were derepressed in the ectoderm and mesoderm butnot in the endoderm (arrows). In A and B, anterior is to the left anddorsal is up. In C, anterior is up and dorsal is to the right. In D toG, dorsal is up.

FIG. 5 (panels A-N) illustrate ventralization of the neural tube inptc^(−/−) embryos. Lateral view of E8.5 wild-type (left) and ptc^(−/−)(right) embryos hybridized with a HNF3b probe. Expression is expandeddorsally in the mutant. (B, C) Transverse sections through the hindbrainof E8.5 wild-type (B) and ptc^(−/−) (C) embryos hybridized with³⁵S-labeled Shh probe (8). Shh is expressed in the floor plate (fp) andnotochord (nc) of the wild-type embryo, and is greatly expanded in theptc mutant. g=gut (D, E) Hematoxylin and eosin stained transversesections through the hindbrain of wild-type (D) and ptc^(−/−) (E) E8.5embryos. Bottle-shaped cells with basal nuclei are indicated by arrows.(F, G) Transverse sections through E8.5 ptc^(+/−) (F) and ptc^(−/−) (G)embryos hybridized with Pax6 probe show loss of expression from the ptcmutant. (H) Dorsal view of E8.25-E8.5 embryos hybridized with Pax3probe. Because of the kinking in the neural tube, the ptc^(−/−) embryois curled on itself. Weak Pax3 expression is seen in the posteriordorsal neural tube of the ptc^(−/−) embryo (bottom, arrow). (I, J)Transverse sections through E8.5 wild-type (I) and ptc^(−/−) (J) embryoshybridized with Pax3 probe. Pax3 is expressed in the dorsal neural tube(nt) and dermamyotome (dm) in the wild-type, but is only present in asmall dorsal domain of the mutant neural tube. s=somite (K, L) Lateralviews of E9 wild type (K) and E8.5 ptc^(−/−) (L) embryos hybridized witherb-b3 probe. Staining is seen in migrating neural crest in the head andsomites of wild type but not mutant embryos (red arrows). Weak stainingin the head, heart and gut (black arrows) is background or non-neuralcrest related. (M) Lateral view of wild type (top) and ptc^(−/−)(bottom) embryos hybridized with Nkx2.1 probe. The body of the mutant istwisted. Nkx2.1 expression is limited to the anterior, but is expandeddorsally in the mutant. (N) Lateral view of E8.5 ptc^(+/−) (left) andptc^(−/−) (right) embryos hybridized with hoxb1 probe. Loss ofexpression in rhombomere four is indicated by the asterisks. In alltransverse sections, dorsal is up. In A, K, L and N, anterior is up anddorsal is to the right. In H and M, anterior is to the left.

FIG. 6 (panels A-F) depict keletal abnormalities and medulloblastomas inptc^(+/−) mice (A) Alcian blue and Alizarin red stained hindlimb from aptc^(+/−) mouse (30). The preaxial digit is duplicated (arrows). (B, C)Dorsal views of brains from wild-type (B) and ptc^(+/−) (C) mice.Anterior is up. In the posterior wild-type brain, the colliculi (col)are present as distinct bumps between the cortex (cor) and cerebellum(ce). In the ptc^(+/−) mouse, a massive medulloblastoma (mb, outlined inred) grew over the colliculi and normal cerebellum, which can no longerbe seen. The olfactory bulbs were removed. (D, E) Hematoxylin and eosinstained section through human (D) and mouse (E) medulloblastomas. Thetumor cells are small with dark, carrot-shaped nuclei (arrows) and formnodules with no apparent orientation. (F) Synaptophysin immunoreactivityin a mouse medulloblastoma (26). Synaptophysin staining (brown) is seenin some processes (arrows). Nuclei are purple.

FIG. 7 (panels A-G) illustrate derepression of ptc and Gli expression inmedulloblastomas from ptc^(+/−) mice. (A to C) Semi-adjacent sectionsthrough a tumor in the cerebellum of a ptc^(+/−) mouse hybridized with³⁵S labeled probes to ptc (A), Gli (B) and Shh (C). ptc and Glitranscripts are abundant in the tumors (asterisks) compared to nearbycerebellar tissue (arrows). No Shh was detected in the tumor. (D)ptc^(+/−) cerebellum (ce) and tumor (mb) stained with X-gal (28).Anterior is to the left. Derepression of ptc expression in themedulloblastoma is reflected in the high level of X-gal staining. (E)Surface staining in (arrows) regions of ptc^(+/−) cerebellum contrastwith absence of b-galactosidase activity in most folia (asterisk). (F)Sagittal section through cerebellum in E. X-gal staining nuclei (arrow)accumulated superficial to the molecular layer (ml), where stainednuclei are not normally seen. In unaffected regions of the cerebellum;X-gal staining was seen in scattered cells of the molecular layer (ml),strongly kin the Purkinje cell layer (pcl) and weakly in the granulecell layer (gl). (G) ptc expression was examined in total RNA (15 mg)from wild-type (WT) and ptc^(+/−) (+/−) cerebellums using a probe (M2-2)(6) that detects exons downstream of the lacZ and neo insertions. ActinmRNA was used as an RNA loading control. The ptc^(+/−) mice had ˜50%decrease in ptc transcripts.

Database References for Nucleotide and Amino Acid Sequences

The sequence for the D. melanogaster patched gene has the Genbankaccession number M28418. The sequence for the mouse patched gene has theGenbank accession number It30589-V46155. The sequence for the humanpatched gene has the Genbank accession number U59464.

DETAILED DESCRIPTION OF THE INVENTION

Vertebrate and invertebrate patched (ptc) gene compositions and methodsfor their isolation are provided. Of particular interest are mammalianptc genes, such as the human and mouse homologs described in theappended examples. The ptc gene, in mammals, is a tumor suppressor anddevelopmental regulator. Certain human cancers, e.g. basal cellcarcinoma, transitional cell carcinoma of the bladder, meningiomas,medulloblastomas, etc., can be characterized by ptc loss-of-function,such as that resulting from oncogenic mutations at the ptc locus, orother loss-of-function mutations which decrease ptc activity in thecell. As described below, we have observed somatic mutations in the ptcgene in a variety of sporadic cancers. For instance, the basal cellnevus syndrome (BCNS), an inherited disorder, is associated withgermline mutations in ptc. Some patients with basal cell nevus syndrome(BCNS) have germ line mutations in ptc, and are at increased risk fordevelopmental defects such as spina bifida and craniofacialabnormalities, basal cell carcinoma (BCC) of the skin, and brain tumors.Mutations to ptc genes are also observed to occur in sporadic BCCs,which generally have both copies of ptc inactivated.

The term “loss-of-function” is art recognized and, with respect to apatched gene or gene product refers to mutations in a patched gene whichultimately decrease or otherwise inhibit the ability of a cell totransduce patched-mediated signals, e.g., the cells may loseresponsiveness to hedgehog induction. For example, a loss-of-functionmutation to a patched gene may be a point mutation, deletion orinsertion of sequences in the coding sequence, intron sequence or 5′ or3′ flanking sequences of the gene so as to, for example, (i) alter(e.g., decrease) the level patched expression, (ii) alter exon-splicingpatterns, (iii) alter the ability of the encoded patched protein tointeract with extracellular or intracellular proteins (such ashedgehog), or (iv) alter (decrease) the stability of the encoded patchedprotein.

The term “aberrant modification” is art recognized and, with respect toa patched gene, refers to a non-wildtype mutation or other alteration tothe gene, e.g., which results in full or partial loss-of-function of thepatched protein or expression of the patched gene.

Such mutations affecting ptc activity have also been associated withother human cancers, including carcinomas, adenocarcinomas, sarcomas andthe like. Decreased ptc activity is also associated with inheriteddevelopmental abnormalities, e.g. rib and craniofacial abnormalities,polydactyly, syndactyly and spina bifida.

The art-recognized term “predisposing mutation”, as it pertains topatched genes, refers to mutations to the patched gene which result inloss-of-function.

The term “genetic predisposition” is art recognized, and refers to agenotype of an animal which predisposes the animal to developing acertain pathological conditions with a frequency (probability) greaterthan the average for the overall population of that animal, taking intoaccount, as appropriate, age, sex or other related physical or medicalcondition(s).

The ptc genes and fragments thereof, encoded protein, and anti-ptcantibodies are useful in the identification of individuals predisposedto development of a variety of cancers and developmental abnormalities,and in characterizing the phenotype of various tumors or otherproliferative or degenerative disorders that are associated with thisgene, e.g., for diagnostic and/or prognostic benefit. Thecharacterization is useful for prenatal screening; and in determiningthe phenotype of a proliferative disorder, e.g. for determining a courseof treatment of the patient. Tumors may be typed or staged as to the ptcstatus, e.g. by detection of mutated sequences, antibody detection ofabnormal protein products, and functional assays for altered ptcactivity.

The terms “developmental disorder” and “develpmental abnormality” areart recognized, and refer to abberant development of a cell, tissue ororgan, e.g., in size, symmetry or functional performance, whichabnormality may or may not be untowardly manifest.

The term “proliferative disorder” is art recognized and refers to adisorder affecting an animal in a manner which is marked by abberant, orotherwise unwanted, proliferation of a subset of cells of an animal.Cancers are proliferative disorders.

The encoded ptc protein is also useful in drug screening forcompositions that mimic ptc activity or expression, including alteredforms of ptc protein, particularly with respect to ptc function as atumor suppressor in oncogenesis.

The human and mouse ptc gene sequences and isolated nucleic acidcompositions are provided in the appended examples. In identifying themouse and human patched genes, cross-hybridization of DNA andamplification primers were employed to move through the evolutionary,tree from the known Drosophila ptc sequence, identifying a number ofinvertebrate homologs.

The human patched gene has been mapped to human chromosome band 9q22.3,and lies between the polymorphic markers D9S196 and D9S287 (a detailedmap of human genome markers may be found in Dib et al. (1996) Nature280:152-http://www.genethon.fr).

As will be understood by those skilled in the art, the method of thepresent invention can be carried out using any of a large number ofassay techniques for detecting alterations in ptc genes and/or ptcprotein function. For instance, individuals are screened by analyzingtheir DNA or RNA for the presence of a predisposing oncogenic ordevelopmental mutation, as compared to a normal sequence. An exemplary“normal” sequence of patched is provided in SEQ ID NO:19 (human).Specific mutations of interest include any mutation that leads tooncogenesis or developmental abnormalities, including insertions,substitutions and deletions in the coding region sequence, in theintrons (e.g., that affect splicing), in the transcriptional regulatorysequences (such as promoter or enhancer sequences) that affect theactivity and expression of the protein.

In general, the subject method can be characterized as including a stepof detecting, in a sample of cells from a patient, the presence orabsence of ptc expression (at the protein or mRNA transcript level),mutations to the ptc gene (coding or non-coding sequence) and/or thefunctional activity of ptc in the sample of cells (such as induction ofGli or the like). Moreover, the subject method can be used to assess thephenotype of cells which are known to be transformed, the phenotyperesults being useful in planning a particular therapeutic regimen.

To illustrate, nucleic acid samples are obtained from a patient having,or suspected as being at risk for developing, a tumor or developmentalabnormality which may be associated with ptc. The nucleic acid isanalyzed for the presence of a predisposing mutation in the ptc gene.The presence of a mutated ptc sequence that affects the level ofexpression of the gene, stability of the gene product, and/or signaltransduction activity of ptc confers an increased susceptibility to aproliferative or developmental disorder. Thus, the level of expressionof ptc can be used predictively to evaluate whether a sample of cellscontains cells which are, or are predisposed towards becoming,transformed.

Diagnostic/prognostic screening of tissue/cell samples for tumors ordevelopmental abnormalities may also be based on the functional orantigenic characteristics of the protein. Immunoassays designed todetect the normal or abnormal ptc protein may be used in screening.Where many diverse mutations lead to a particular disease phenotype,functional protein assays have proven to be effective screening tools.Such assays may be based on detecting changes in the transcriptionalregulation mediated by ptc, or may directly detect ptc activities suchas hedgehog binding, transporter activity or the like, or may involveantibody localization of patched in cells.

Inheritance of BCNS is autosomal dominant, although many cases are theresult of new mutations. Diagnosis of BCNS is performed by protein, DNAsequence or hybridization analysis of any convenient sample from apatient, e.g. biopsy material, blood sample, scrapings from cheek, etc.A typical patient genotype will have a predisposing mutation on at leastone chromosome. In tumors and at least sometimes developmentallyaffected tissues, loss of heterozygosity at the ptc locus leads toaberrant cell and tissue behavior. When the normal copy of ptc is lost,leaving only the reduced function mutant copy, abnormal cell growth andreduced cell layer adhesion is the result. Examples of specific ptcmutations in BCNS patients are a 9 bp insertion at nt 2445 of the codingsequence- and an 11 bp deletion of nt 2441 to 2452 of the codingsequence. These result in insertions or deletions in the region of theseventh transmembrane domain.

Prenatal diagnosis of BCNS may be performed, particularly where there isa family history of the disease, e.g. an affected parent or sibling. Itis desirable, although not required, in such cases to determine thespecific predisposing mutation present in affected family members. Asample of fetal DNA, such as an amniocentesis sample, fetal nucleated orwhite blood cells isolated from maternal blood, chorionic villus sample,etc. is analyzed for the presence of the predisposing mutation.Alternatively, a protein based assay, e.g. functional assay orimmunoassay, is performed on fetal cells known to express ptc.

Sporadic tumors associated with loss of ptc function include a number ofcarcinomas and other transformed cells known to have deletions in theregion of chromosome 9q22, e.g. basal cell carcinomas, transitionalbladder cell carcinoma, meningiomas, medullomas, fibromas of the heartand ovary, and carcinomas of the lung, ovary, kidney and esophagus.Characterization of sporadic tumors will generally require analysis oftumor cell DNA, conveniently with a biopsy sample. A wide range ofmutations are found in sporadic cases, up to and including deletion ofthe entire long arm of chromosome 9. Oncogenic mutations may delete oneor more exons, e.g. 8 and 9, may affect the amino acid sequence such asof the extracellular loops or transmembrane domains, may causetruncation of the protein by introducing a frameshift or stop codon,etc. Specific examples of oncogenic mutations include a C to Ttransition at nt 523 and deletions encompassing exon 9. C to Ttransitions are characteristic of ultraviolet mutagenesis, as expectedwith cases of skin cancer.

Biochemical studies may be performed to determine whether a candidatesequence variation in the ptc coding region or control regions isoncogenic. For example, a change in the promoter or enhancer sequencethat downregulates expression of patched may result in predisposition tocancer. Expression levels of a candidate variant allele are compared toexpression levels of the normal allele by various methods known in theart. Methods for determining promoter or enhancer strength includequantitation of the expressed mRNA or ptc protein; insertion of thevariant control element into a vector with a reporter gene such asβ-galactosidase, chloramphenical acetyltransferase, etc. that providesfor convenient quantitation- and the like. Nuclear run-off assays areanpther convenient means for measuring promoter/enhancer activity. Theactivity of the encoded ptc protein may be determined by comparison withthe wild-type protein, e.g. by detection of transcriptional regulationof TGF or Wnt family genes, Gli genes, ptc itself, or reporter genefusions involving transcriptional regulatory sequences of these targetgenes.

The term “patched-dependent gene”, or “a gene which is regulated in apatched-dependent manner”, refers to genes, such as Gli or patched, etc,whose level of expression is regulated at least in part by the presenceof a patched protein in the cell, e.g., can be controlled bypatched-dependent intracellular signals.

A human patched gene (SEQ ID NO:18) has a 4.5 kb open reading frameencoding a protein of 1447 amino acids. Including coding and noncodingsequences, it is about 89% identical at the nucleotide level to themouse patched gene (SEQ ID NO:9). A mouse patched gene (SEQ ID NO:9)encodes a protein (SEO ID NO:10) that has about 38% identical aminoacids to Drosophila ptc (SEQ ID NO:6), over about 1,200 amino acids. Thebutterfly homolog (SEQ ID NO:4) is 1,300 amino acids long and overallhas a 50% amino acid identity to fly ptc (SEQ ID NO:6). A 267 bp exonfrom the beetle patched gene encodes an 89 amino acid protein fragment,which was found to be 44% and 51% identical to the corresponding regionsof fly and butterfly ptc respectively.

The DNA sequence encoding ptc may be cDNA, RNA, genomic DNA orsynthetic, an includes fragments of the full-length coding sequence. Theterm “patched gene” shall be intended to mean the open reading frameencoding specific ptc polypeptides, as well as, as appropriate, adjacentintronic sequences and 5′ and 3′ non-coding nucleotide sequencesinvolved in the regulation of expression, up to about 1 kb beyond thecoding region, in either direction. The gene may be introduced into anappropriate vector for extrachromosomal maintenance or for integrationinto the host.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons, 3′ and 5′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns deleted, to create a continuous open reading frameencoding ptc.

The genomic ptc sequence has a non-contiguous open reading frame, whereintrons interrupt the coding regions. A genomic sequence of interestcomprises the nucleic acid present between the initiation codon and thestop codon, as defined in the listed sequences, including all of theintrons that are normally present in a native chromosome. It may furtherinclude the 3′ and 5′ untranslated regions found in the mature mRNA. Itmay further include specific transcriptional and translationalregulatory sequences, such as promoters, enhancers, etc., includingabout 1 kb of flanking genomic DNA at either the 5′ or 3′ end of thecoding region. The genomic DNA may be isolated as a fragment of 50 kbpor smaller; and substantially free of flanking chromosomal sequence.

The nucleic acid compositions of the subject invention encode all or apart of the subject polypeptides. Fragments may be obtained of the DNAsequence by chemically synthesizing oligonucleotides in accordance withconventional methods, by restriction enzyme digestion, by PCRamplification, etc. For the most part, DNA fragments will be of at least15 nt, usually at least 18 nt, more usually at least about 50 nt. Suchsmall DNA fragments are useful as primers for PCR, hybridizationscreening, etc. Larger DNA fragments, i.e. greater than 100 nt areuseful for production of the encoded polypeptide. For use inamplification reactions, such as PCR, a pair of primers will be used.The exact composition of the primer sequences is not critical to theinvention, but for most applications the primers will hybridize to thesubject sequence under stringent conditions, as known in the art. It ispreferable to chose a pair of primers that will generate anamplification product of at least about 50 nt, preferably at least about100 nt. Algorithms for the selection of primer sequences are generallyknown, and are available in commercial software packages. Amplificationprimers hybridize to complementary strands of DNA, and will primetowards each other.

A vareity of techniques are know in the art for generating fragments ofthe subject patched proteins, particularly those which retain theability to bind hedgehog and/or alter the level of expression of apatched-dependent gene when expressed in a cell. To further illustratethe state of the art of combinatorial mutagenesis, it is noted that thereview article of Gallop et al. (1994) J Med Chem 37:1233 describes thegeneral state of the art of combinatorial libraries as of the earlier1990's. In particular, Gallop et al state at page 1239 “[s]creening theanalog libraries aids in determining the minimum size of the activesequence and in identifying those residues critical for binding andintolerant of substitution”. In addition, the Ladner et al. PCTpublication WO90/02809, the Goeddel et al. U.S. Pat. No. 5,223,408, andthe Markland et al. PCT publication WO92/15679 illustrate specifictechniques which one skilled in the art could utilize to generatelibraries of patched variants which can be rapidly screened to identifyvariants/fragments which retained a particular activity, such as signaltransduction (measured by transcriptional regulation)and/or the abilityto bind hedgehog polypeptides. These techniques are exemplary of the artand demonstrate that large libraries of related variants/truncants canbe generated and assayed to isolate particular variants without undueexperimentation. Gustin et al. (1993) Virology 193:653, and Bass et al.(1990) Proteins: Structure, Function and Genetics 8:309-314 alsodescribe other exemplary techniques from the art which can be adapted asmeans for generating mutagenic variants of patched polypeptides.

Indeed, it is plain from the combinatorial mutagenesis art that largescale mutagenesis of patched proteins, without any preconceived ideas ofwhich residues were critical to the biological function, and generatewide arrays of variants having equivalent biological activity. In fact,it is the ability of combinatorial techniques to screen billions ofdifferent variants by high throughout analysis that removes anyrequirement of a priori understanding or knowledge of critical residues.

To illustrate, the amino acid sequences for a population of patchedhomologs or other related proteins are aligned, preferably to promotethe highest homology possible. Such a population of variants caninclude, for example, the subject patched homologs from variousvertebrate and invertebrate species. Amino acids which appear at eachposition of the aligned sequences are selected to create a degenerateset of combinatorial sequences. In a preferred embodiment, thevariegated library of patched variants is generated by combinatorialmutagenesis at the nucleic acid level, and is encoded by a variegatedgene library. Such techniques have been employed in the directedevolution of other proteins (see, for example, Scott et al. (1990)Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin etal. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point or tuncationmutations, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of patched homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. For example, simple binding assayscan be performed with hedgehog protein. In other embodiments, theability of the library gene to alter the transcriptional rate of areporter gene can be detected. Each of such illustrative assays areamenable to high through-put analysis as necessary to screen largenumbers of degenerate patched sequences created by combinatorialmutagenesis techniques.

Combinatorial mutagenesis has a potential to generate very largelibraries of mutant proteins, e.g., in the order of 10²⁶ molecules.Combinatorial libraries of this size may be technically challenging toscreen even with high throughput screening assays such as phage display.To overcome this problem, a new technique has been developed recently,recrusive ensemble mutagenesis (REM), which allows one to avoid the veryhigh proportion of non-functional proteins in a random library andsimply enhances the frequency of functional proteins, thus decreasingthe complexity required to achieve a useful sampling of sequence space.REM is an algorithm which enhances the frequency of functional mutantsin a library when an appropriate selection or screening method isemployed (Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan etal., 1992, Parallel Problem Solving from Nature, 2., In Maenner andManderick, eds., Elsevir Publishing Co., Amsterdam, pp. 401-410;Delgrave et al., 1993, Protein Engineering 6(3):327-331).

In prefered embodiments, the subject patched protein is a fragment ofthe full length protein which binds to hedgehog polypeptides, e.g.,including one or both of the substantial extracellular (hydrohilic)domains, such as corresponding to residues Asn120-Ser438 and/orArg770-Trp1027 of the human patched protein. Other preferred embodimentsof patched polypeptides include fragments which are at least 50, 75,100, 150 and 200 amino acid residues in length. Such forms of thepatched protein can be provided in soluble form, as for example apreparation of one of the extracellular domains, or a preparation ofboth of the extracellular domains which are covalently connected by anunstructured linker (see, for example, Huston et al. (1988) PNAS85:4879; and U.S. Pat. No. 5,091,513). In other embodiments, the proteincan be provided as part of a liposomal preparation or expressed on thesurface of a cell.

In yet other preferred embodiments, the patched polypeptide is at least60, 65, 75, 85, 90, or 95 percent identical with the human or mousepatched sequences of the appended sequence listing.

The ptc genes are isolated and obtained in substantial purity, generallyas other than an intact mammalian chromosome. Usually, the DNA will beobtained substantially free of other nucleic acid sequences that do notinclude a ptc sequence or fragment thereof, generally being at leastabout 50%, usually at least about 90% pure and are typically“recombinant”, i.e. flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome.

The DNA sequences are used in a variety of ways. They may be used asprobes for identifying other patched genes. Mammalian homologs havesubstantial sequence similarity to the subject sequences, i.e. at least75%, usually at least 90%, more usually at least 95% sequence identitywith the nucleotide sequence of the subject DNA sequence. Sequencesimilarity is calculated based on a reference sequence, which may be asubset of a larger sequence, such as a conserved motif, coding region,flanking region, etc. A reference sequence will usually be at leastabout 18 nt long, more usually at least about 30 nt long, and may extendto the complete sequence that is being compared. Algorithms for sequenceanalysis are known in the art, such as BLAST, described in Altschul etal. (1990) J Mol Biol 215:403-10.

Nucleic acids having sequence similarity are detected by hybridizationunder low stringency conditions, for example, at 50 C and 10×SSC (0-9 Msaline/0.09 M sodium citrate) and remain bound when subjected to washingat 55° C. in 1×SSC. By using probes, particularly labeled probes of DNAsequences, one can isolate homologous or related genes. The source ofhomologous genes may be any mammalian species, e.g. primate species,particularly human-murines, such as rats and mice, canines, felines,bovines, ovines, equines, etc.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell-established in the literature and does not require elaborationhere. Conveniently, a biological specimen is used as a source of mRNA.The mRNA may be amplified by RT-PCR, using reverse transcriptase to forma complementary DNA strand, followed by polymerase chain reactionamplification using primers specific for the subject DNA sequences.Alternatively, the mRNA sample is separated by gel electrophoresis,transferred to a suitable support, e.g., nitrocellulose and then probedwith a fragment of the subject DNA as a probe. Other techniques may alsofind use. Detection of mRNA having the subject sequence is indicative ofpatched gene expression in the sample.

The subject nucleic acid sequences may be modified for a number ofpurposes, particularly where they will be used intracellularly, forexample, by being joined to a nucleic acid cleaving agent, e.g. achelated metal ion, such as iron or chromium for cleavage of the gene;as an antisense sequence, or the like. Modifications may includereplacing oxygen of the phosphate esters with sulfur or nitrogen,replacing the phosphate with phosphoramide, etc.

A number of methods are available for analyzing genomic DNA sequences.Where large amounts of DNA are available, the genomic DNA is useddirectly. Alternatively, the region of interest is cloned into asuitable vector and grown in sufficient quantity for analysis, oramplified by conventional techniques, such as the polymerase chainreaction (PCR). The use of the polymerase chain reaction is described inSaiki, et al. (1985) Science 239:487, and a review of current techniquesmay be found in Sambrook, et al. Molecular Cloning: A Laboratory Manual,CSH Press 1989, pp.14.2-14.33.

A detectable label may be included in the amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-Xrhodamine (ROX), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein(HEX), 5-carboxyfluorescein (5-FAM) orN,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels,e.g. 32P, 35S, 3H; etc. The label may be a two stage system, where theamplified DNA is conjugated to biotin, haptens, etc. having a highaffinity binding partner, e.g. avidin, specific antibodies, etc., wherethe binding partner is conjugated to a detectable label. The label maybe conjugated to one or both of the primers. Alternatively, the pool ofnucleotides used in the amplification is labeled, so as to incorporatethe label Into the amplification product.

The amplified or cloned fragment may be sequenced by dideoxy or othermethods, and the sequence of bases compared to the normal ptc sequence.Hybridization with the variant sequence may also be used to determineits presence, by Southern blots, dot blots, etc. Single strandconformational polymorphism (SSCP) analysis, denaturing gradient gelelectrophoresis (DGGE), and heteroduplex analysis in gel matrices areused to detect conformational changes created by DNA sequence variationas alterations in electrophoretic mobility. The hybridization pattern ofa control and variant sequence to an array of oligonucleotide probesimmobilized on a solid support, as described in WO 95/11995, may also beused as a means of detecting the presence of variant sequences.Alternatively, where a predisposing mutation creates or destroys arecognition site for a restriction endonuclease, the fragment isdigested with that endonuclease, and the products size fractionated todetermine whether the fragment was digested. Fractionation is performedby gel electrophoresis, particularly acrylamide or agarose gels.

In a merely illustrative embodiment, the method includes the steps of(i) collecting a sample of cells from a patient, (ii) isolating nucleicacid (e.g., genomic, mRNA or both) from the cells of the sample, (iii)contacting the nucleic acid sample with one or more primers whichspecifically hybridize to a ptc gene under conditions such thathybridization and amplification of the ptc gene (if present) occurs, and(iv) detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample.

In yet another exemplary embodiment, aberrant methylation patterns of aptc gene can be detected by digesting genomic DNA from a patient samplewith one or more restriction endonucleases that are sensitive tomethylation and for which recognition sites exist in the ptc gene(including in the flanking and intronic sequences). See, for example,Buiting et al., (1994) Human Mol Genet 3:893-895. Digested DNA isseparated by gel electrophoresis, and hybridized with probes derivedfrom, for example, genomic or cDNA sequences. The methylation status ofthe ptc gene can be determined by comparison of the restriction patterngenerated from the sample DNA with that for a standard of knownmethylation.

In still another embodiment a diagnostic assay is provided which detectsthe ability of a ptc gene product, e.g., recombinantly expressed from agene isolated from a biopsied cell, to bind to other proteins, e.g.,upstream (hedgehog) or downstream of ptc. For instance, it will bedesirable to detect ptc mutants which bind with lower binding affinityfor hedgehog proteins. Such mutants may arise, for example, from finemutations, e.g., point mutants, which may be impractical to detect bythe diagnostic DNA sequencing techniques or by the immunoassaysdescribed above. The present invention accordingly further contemplatesdiagnostic screening assays which generally comprise cloning one or moreptc genes from the sample cells, and expressing the cloned genes underconditions which permit detection of an interaction between thatrecombinant gene product and a ptc-binding protein, e.g., a hedgehogprotein. As will be apparent from the description of the various drugscreening assays set forth below, a wide variety of techniques can beused to determine the ability of a ptc protein to bind to other cellularcomponents.

The subject nucleic acids can be used to generate transgenic animals orsite specific gene modifications in cell lines. Transgenic animals maybe made through homologous recombination, where the normal patched locusis altered. Alternatively, a nucleic acid construct is randomlyintegrated into the genome, Vectors for stable integration includeplasmids, retroviruses and other animal viruses, YACS, and the like.

The modified cells or animals are useful in the study of patchedfunction and regulation. For example, a series of small deletions and/orsubstitutions may be made in the patched gene to determine the role ofdifferent exons in oncogenesis, signal transduction, etc. Of particularinterest are transgenic animal models for carcinomas of the skin, whereexpression of ptc is specifically reduced or absent in skin cells. Analternative approach to transgenic models for this disease are thosewhere one of the mammalian hedgehog genes, e.g. Shh, lhh, Dhh, areupregulated in skin cells, or in other cell types. For models of skinabnormalities, one may use a skin-specific promoter to drive expressionof the transgene, or other inducible promoter that can be regulated inthe animal model. Such promoters include keratin gene promoters.Specific constructs of interest include anti-sense ptc, which will blockptc expression, expression of dominant negative ptc mutations, andover-expression of HH genes. A detectable marker, such as lacZ may beintroduced into the patched locus, where upregulation of patchedexpression will result in an easily detected change in phenotype.

One may also provide for expression of the patched gene or variantsthereof in cells or tissues where it is not normally expressed or atabnormal times of development. Thus, mouse models of spina bifida orabnormal motor neuron differentiation in the developing spinal cord aremade available. In addition, by providing expression of ptc protein incells in which it is otherwise not normally produced, one can inducechanges in cell behavior, e.g. through ptc mediated transcriptionmodulation.

DNA constructs for homologous recombination will comprise at least aportion of the patched or hedgehog gene with the desired geneticmodification, and will include regions of homology to the target locus.DNA constructs for random integration need not include regions ofhomology to mediate recombination. Conveniently, markers for positiveand negative selection are included. Methods for generating cells havingtargeted gene modifications through homologous recombination are knownin the art. For various techniques for transfecting mammalian cells, seeKeown et al. (1990) Methods in Enzymology 185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, or EScells may be obtained freshly from a host, e.g. mouse, rat, guinea pig,etc. Such cells are grown on an appropriate fibroblast-feeder layer orgrown in the presence of leukemia inhibiting factor (LIF). When ES cellshave been transformed, they may be used to produce transgenic animals.After transformation, the cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be detected byemploying a selective medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination or integration of the construct. Those colonies that arepositive may then be used for embryo manipulation and blastocystinjection. Blastocysts are obtained from 4 to 6 week old superovulatedfemales. The ES cells are trypsinized, and the modified cells areinjected into the blastocoel of the blastocyst. After injection, theblastocysts are returned to each uterine horn of pseudopregnant females.Females are then allowed to go to term and the resulting littersscreened for mutant cells having the construct. By providing for adifferent phenotype of the blastocyst and the ES cells, chimeric progenycan be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic grafts or transplants, or in in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc., e.g. to determine the effectof a candidate drug on basal cell carcinomas.

The subject gene may be employed for producing all or portions of thepatched protein. For expression, an expression cassette may be employed,providing for a transcriptional and translational initiation region,which may be, inducible or constitutive, the coding region under thetranscriptional control of the transcriptional initiation region, and atranscriptional and translational termination region. Varioustranscriptional initiation regions may be employed which are functionalin the expression host.

Specific ptc peptides of interest include the extracellular domains,particularly in, the human mature protein, aa 120 to 437, and aa 770 to1027. These peptides may be used as immunogens to raise antibodies thatrecognize the protein in an intact cell membrane. The cytoplasmicdomains, as shown in FIG. 2, (the amino terminus and carboxy terminus)are of interest in binding assays to detect ligands involved insignaling mediated by ptc.

The peptide may be expressed in prokaryotes or eukaryotes in accordancewith conventional ways, depending upon the purpose for expression. Forlarge scale production of the protein, a unicellular organism or cellsof a higher organism, e.g. eukaryotes such as vertebrates, particularlymammals, may be used as the expression host, such as E. coli, B,subthis, S. cerevisiae, and the like. In many situations, it may bedesirable to express the patched gene in a mammalian host, whereby thepatched gene will be glycosylated, and transported to the cellularmembrane for various studies.

With the availability of the protein in large amounts by employing anexpression host, the protein may be isolated and purified in accordancewith conventional ways. A lysate may be prepared of the expression hostand the lysate purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography; or other purificationtechnique. The purified protein will generally be at least about 80%pure, preferably at least about 90% pure, and may be up to and including100% pure. By pure is intended free of other proteins, as well ascellular debris.

The polypeptide is used for the production of antibodies, where shortfragments provide for antibodies specific for the particularpolypeptide, whereas larger fragments or the entire gene allow for theproduction of antibodies over the surface of the polypeptide or protein.Antibodies may be raised to the normal or mutated forms of ptc- Theextracellular domains of the protein are of interest as epitopes,particular antibodies that recognize common changes found in abnormal,oncogenic ptc, which compromise the protein activity. Antibodies may beraised to isolated peptides corresponding to these domains, or to thenative protein, e.g. by immunization with cells expressing ptc,immunization with liposomes having ptc inserted in the membrane, etc.Antibodies that recognize the extracellular domains of ptc are useful indiagnosis, typing and staging of human carcinomas.

Antibodies are prepared in accordance with conventional ways, where theexpressed polypeptide or protein may be used as an immunogen, by itselfor conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg,other viral or eukaryotic proteins, or the like. Various adjuvants maybe employed, with a series of injections, as appropriate, For monoclonalantibodies, after one or more booster injections, the spleen may beisolated, the splenocytes immortalized, and then screened for highaffinity antibody binding. The immortalized cells, e.g. hybridomas,producing the desired antibodies may then be expanded. For furtherdescription, see Monoclonal Antibodies- A Laboratory Manual, Harlow andLane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,1988. If desired, the mRNA encoding the heavy and light chains may beisolated and mutagenized by cloning in E. coli, and the heavy and lightchains may be mixed to further enhance the affinity of the antibody.

The antibodies find particular use in diagnostic assays fordevelopmental abnormalities, basal cell carcinomas and other tumorsassociated with mutations in ptc. Staging, detection and typing oftumors may utilize a quantitative immunoassay for the presence orabsence of normal ptc. Alternatively, the presence of mutated forms ofptc may be determined. A reduction in normal ptc and/or presence ofabnormal ptc is indicative that the tumor is ptc-associated.

A sample is taken from a patient suspected of having a ptc-associatedtumor, developmental abnormality or BCNS. Samples, as used herein,include biological fluids such as blood, cerebrospinal fluid, tears,saliva, lymph, dialysis fluid and the like organ or tissue culturederived fluids, and fluids extracted from physiological tissues. Alsoincluded in the term are derivatives and fractions of such fluids.Biopsy samples are of particular interest, e.g. skin lesions, organtissue fragments, etc. Where metastasis is suspected, blood samples maybe preferred. The number of cells in a sample will generally be at leastabout 10³, usually at least 10⁴ more usually at least about 10⁵. Thecells may be dissociated, in the case of solid tissues, or tissuesections may be analyzed. Alternatively a lysate of the cells may beprepared.

Diagnosis may be performed by a number of methods. The different methodsall determine the absence or presence of normal or abnormal ptc inpatient cells suspected of having a mutation in ptc. For example,detection may utilize staining of intact cells or histological sections,performed in accordance with conventional methods. The antibodies ofinterest are added to the cell sample, and incubated for a period oftime sufficient to allow binding to the epitope, usually at least about10 minutes. The antibody may be labeled with radioisotopes, enzymes,fluorescers, chemiluminescers, or other labels for direct detection.Alternatively, a second stage antibody or reagent is used to amplify thesignal. Such reagents are well-known in the art. For example, theprimary antibody may be conjugated to biotin, with horseradishperoxidase-conjugated avidin added as a second stage reagent. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

An alternative method for diagnosis depends on the in vitro detection ofbinding between antibodies and ptc in a lysate. Measuring theconcentration of ptc binding in a, sample or fraction thereof may beaccomplished by a variety of specific assays. A conventional sandwichtype assay may be used. For example, a sandwich assay may first attachptc-specific antibodies to an insoluble surface or support. Theparticular manner of binding is not crucial so long as it is compatiblewith the reagents and overall methods of the invention They may be boundto the plates covalently or non-covalently, preferably non-covalently.

The insoluble supports may be any compositions to which polypeptides canbe bound, which is readily separated from soluble material, and which isotherwise compatible with the overall method. The surface of suchsupports may be solid or porous and of any convenient shape. Examples ofsuitable insoluble supports to which the receptor is bound includebeads, e.g. magnetic beads, membranes and microtiter plates. These aretypically made of glass, plastic (e.g. polystyrene), polysaccharides,nylon or nitrocellulose. Microtiter plates are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

Patient sample lysates are then added to separately assayable supports(for example, separate wells of a microtiter plate) containingantibodies. Preferably, a series of standards, containing knownconcentrations of normal and/or abnormal ptc is assayed in parallel withthe samples or aliquots thereof to serve as controls. Preferably, eachsample and standard will be added to multiple wells so that mean valuescan be obtained for each. The incubation time should be sufficient forbinding, generally, from about 0.1 to 3 hr is sufficient. Afterincubation, the insoluble support is generally washed of non-boundcomponents. Generally, a dilute non-ionic detergent medium at anappropriate pH, generally 7-8, is used as a wash medium. From one to sixwashes may be employed, with sufficient volume to thoroughly washnonspecifically bound proteins present in the sample.

After washing, a solution containing a second antibody is applied. Theantibody will bind ptc with sufficient specificity such that it, can bedistinguished from other components present. The second antibodies maybe labeled to facilitate direct, or indirect quantification of binding.Examples of labels that permit direct measurement of second receptorbinding include radiolabels, such as ³H or ¹²⁵I, fluorescers, dyes,beads, chemilumninescers, colloidal particles, and the like. Examples oflabels which permit indirect measurement of binding include enzymeswhere the substrate may provide for a colored or fluorescent product. Ina preferred embodiment, the antibodies are labeled with a covalentlybound enzyme capable of providing a detectable product signal afteraddition of suitable substrate. Examples of suitable enzymes for use inconjugates include horseradish peroxidase, alkaline phosphatase, malatedehydrogenase and the like. Where not commercially available, suchantibody-enzyme conjugates are readily produced by techniques known tothose skilled in the art. The incubation time should be sufficient forthe labeled ligand to bind available molecules. Generally, from about0.1 to 3 hr is sufficient, usually 1 hr sufficing.

After the second binding step, the insoluble support is again washedfree of non-specifically bound material. The signal produced by thebound conjugate is detected by conventional means. Where an enzymeconjugate is used, an appropriate enzyme substrate is provided so adetectable product is formed.

Other immunoassays are known in the art and may find use as diagnostics.Ouchterlony plates provide a simple determination of antibody binding.Western blots may be performed on protein gels or protein spots onfilters, using a detection system specific for ptc as desired,conveniently using a labeling method as described for the sandwichassay.

Other diagnostic assays of interest are based on the functionalproperties of ptc protein itself. Such assays are particularly usefulwhere a large number of different sequence changes lead to a commonphenotype, i.e., loss of protein function leading to oncogenesis ordevelopmental abnormality. For example, a functional assay may be basedon the transcriptional changes mediated by hedgehog and patched geneproducts. Addition of soluble Hh to embryonic stem cells causesinduction of transcription in target genes. The presence of functionalptc can be determined by its ability to antagonize Hh activity. Otherfunctional assays may detect the transport of specific moleculesmediated by ptc, in an intact cell or membrane fragment. Conveniently, alabeled substrate is used, where the transport in or out of the cell canbe quantitated by radiography, microscopy, flow cytometry,spectrophotometry, etc. Other assays may detect conformational changes,or changes in the subcellular localization of patched protein.

By providing for the production of large amounts of patched protein, onecan identify ligands or substrates that bind to, modulate or mimic theaction of patched A common feature in basal cell carcinoma is the lossof adhesion between epidermal and dermal layers, indicating a role forptc in maintaining appropriate cell adhesion. Areas of investigationinclude the development of cancer treatments, wound healing, adverseeffects of aging, metastasis, etc.

Drug screening identifies agents that provide a replacement for ptcfunction in abnormal cells. The role of ptc as a tumor suppressorindicates that agents which mimic its function, in terms oftransmembrane transport of molecules, transcriptional down-regulation,etc., will inhibit the process of oncogenesis. These agents may alsopromote appropriate cell adhesion in wound healing and aging, to reversethe loss of adhesion observed in metastasis, etc. Conversely, agentsthat reverse ptc function may stimulate controlled growth and healing.Of particular interest are screening assays for agents that have a lowtoxicity for human cells. A wide variety of assays may be used for thispurpose, including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,and the like. The purified protein may also be used for determination ofthree-dimensional crystal structure, which can be used., for modelingintermolecular interactions, transporter function, etc.

The term “agent” as used herein describes any molecule, e.g. protein orpharmaceutical, with the capability of altering or mimicking thephysiological function of patched Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or a combinationsthereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. that are used to facilitate optimal protein-proteinbinding and/or reduce nonspecific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4° and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

Other assays of interest detect agents that mimic patched function, suchas repression of target gene transcription, transport of patchedsubstrate compounds, etc. For example, an expression constructcomprising a patched gene may be introduced into a cell line underconditions that allow expression. The level of patched activity isdetermined by a functional assay, as previously described. In onescreening assay, candidate agents are added in combination with a Hhprotein, and the ability to overcome Hh antagonism of ptc is detected.In another assay, the ability of candidate agents to enhance ptcfunction is determined. Alternatively, candidate agents are added to acell that lacks functional ptc, and screened for the ability toreproduce ptc in a functional assay.

In one embodiment, the drug screening assay is a cell-based assay whichdetects the ability of a compound to alter patched-dependent genetranscription. By selecting transcriptional regulatory sequences fromgenes whose expression is regulated by patched signal transduction, e.g.from patched, GLI, hedgehog or PTHrP genes, e.g., regulatory sequencesthat are responsible for the up- or down regulation of these genes inresponse to patched signalling, and operatively linking such promotersto a reporter gene, one can derive a transcription based assay which issensitive to the ability of a specific test compound to modify patchedsignalling pathways. Expression of the reporter gene, thus, provides avaluable screening tool for the development of compounds that act asagonists or antagonists of patched.

Reporter gene based assays of this invention measure the end stage ofthe above described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on ptc signaling. To identify potentialregulatory elements responsive to ptc signaling present in thetranscriptional regulatory sequence of a target gene, nested deletionsof genomic clones of the target gene can be constructed using standardtechniques. See, for example, Current Protocols in Molecular Biology,Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989); U.S.Pat. No. 5,266,488; Sato et al. (1995) J Biol Chem 270:10314-10322; andKube et al. (1995) Cytokine 7:1-7. A nested set of DNA fragments fromthe gene's 5′-flanking region are placed upstream of a reporter gene,such as the luciferase gene, and assayed for their ability to directreporter gene expression in patched expressing cells. Host cellstransiently transfected with reporter-gene constructs can be scored forthe induction of expression of the reporter gene in the presence andabsence of hedgehog to determine regulatory sequences which areresponsice to patched-dependent signalling.

In practicing one embodiment of the assay, a reporter gene construct isinserted into the reagent cell in order to generate a detection signaldependent on second messengers generated by induction with hedgehogprotein. Typically, the reporter gene construct will include a reportergene in operative linkage with one or more transcriptional regulatoryelements responsive to the hedgehog activity, with the level ofexpression of the reporter gene providing the hedgehog-dependentdetection signal. The amount of transcription from the reporter gene maybe measured using any method known to those of skill in the art to besuitable. For example, mRNA expression from the reporter gene may bedetected using RNAse protection or RNA-based PCR, or the protein productof the reporter gene may be identified by a characteristic stain or anintrinsic activity. The amount of expression from the reporter gene isthen compared to the amount of expression in either the same cell in theabsence of the test compound (or hedgehog) or it may be compared withthe amount of transcription in a substantially identical cell that lacksthe target receptor protein. Any statistically or otherwise significantdifference in the amount of transcription indicates that the testcompound has in some manner altered the signal transduction of thepatched protein, e.g., the test compound is a potential ptc therapeutic.

As described in further detail below, in preferred embodiments the geneproduct of the reporter, is detected by an intrinsic activity associatedwith that product. For instance, the reporter gene may encode a geneproduct that, by enzymatic activity, gives rise to a detection signalbased on color, fluorescence, or luminescence. In other preferredembodiments, the reporter or marker gene provides a selective growthadvantage, e.g., the reporter gene may enhance cell viability, relieve acell nutritional requirement, and/or provide resistance to a drug.

Preferred reporter genes are those that are readily detectable. Thereporter gene may also be included in the construct in the form of afusion gene with a gene that includes desired transcriptional regulatorysequences or exhibits other desirable properties. Examples of reportergenes include, but are not limited to CAT (chloramphenicol acetyltransferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase,and other enzyme detection systems, such as beta-galactosidase; fireflyluciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterialluciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwinet al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh etal. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol.Appl. Gen. 2: 101), human placental secreted alkaline phosphatase(Cullen and Malim (1992).Methods in Enzymol. 216:362-368).

Transcriptional control elements which may be included in a reportergene construct include, but are not limited to, promoters, enhancers,and repressor and activator binding sites. Suitable transcriptionalregulatory elements may be derived from the transcriptional regulatoryregions of genes whose expression is induced after modulation of apatched signal transduction pathway. The characteristics of preferredgenes from which the transcriptional control elements are derivedinclude, but are not limited to, low or undetectable expression inquiescent cells, rapid induction at the transcriptional level withinminutes of extracellular simulation, induction that is transient andindependent of new protein synthesis, subsequent shut-off oftranscription requires new protein synthesis, and mRNAs transcribed fromthese genes have a short half-life. It is not necessary for all of theseproperties to be present.

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of cancer or developmental abnormalities attributable to adefect in patched function. The compounds may also be used to enhancepatched function in wound healing, aging, etc. The inhibitory agents maybe administered in a variety of ways, orally, topically, parenterallye.g. subcutaneously, intraperitoneally, by viral infection,intravascularly, etc. Topical treatments are of particular interest.Depending upon the manner of introduction, the compounds may beformulated in a variety of ways. The concentration of therapeuticallyactive compound in the formulation may vary from about 0.1-100 wt. %.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

The gene or fragments thereof may be used as probes for identifying the5′ non-coding region comprising the transcriptional initiation region,particularly the enhancer regulating the transcription of patched. Byprobing a genomic library, particularly with a probe comprising the 5′coding region, one can obtain fragments comprising the 5′ non-codingregion. If necessary, one may walk the fragment to obtain further 5′sequence to ensure that one has at least a functional portion of theenhancer. It is found that the enhancer is proximal to the 5′ codingregion, a portion being in the transcribed sequence and downstream fromthe promoter sequences. The transcriptional initiation region may beused for many purposes, studying embryonic development, providing forregulated expression of patched protein or other protein of interestduring embryonic development or thereafter, and in gene therapy.

The gene may also be used for gene therapy. Vectors useful forintroduction of the gene include plasmids and viral vectors. Ofparticular interest are retroviral-based vectors, e.g. moloney murineleukemia virus and modified human immunodeficiency virus-adenovirusvectors, etc. Gene therapy may be used to treat skin lesions, anaffected fetus, etc., by transfection of the normal gene into embryonicstem cells or into other fetal cells. A wide variety of viral vectorscan be employed for transfection and stable integration of the gene intothe genome of the cells. Alternatively, micro-injection may be employed,fusion, or the like for introduction of genes into a suitable host cell.See, for example, Dhawan et al. (1991) Science 254:1509-1512 and Smithet al. (1990) Molecular and Cellular Biology 3268-3271.

The following examples are offered by illustration not by way oflimitation.

Experimental

Methods and Materials

PCR on Mosquito (Anopheles gambiae) Genomic DNA. PCR primers were basedon amino acid stretches of fly ptc that were not likely to diverge overevolutionary time and were of low degeneracy. Two such primers (P2R1(SEO ID NO-14)-GGACGAATTCAARGTNCAYCARYTNTGG, P4R1: (SEQ ID NO:15)GGACGAATTCCYTCCCARAARCANTC, (the underlined sequences are Eco RIlinkers) amplified an appropriately sized band from mosquito genomic DNAusing the PCR. The program conditions were as follows:

-   -   94 C 4 min.; 72 C Add Taq;    -   [49 C 30 sec.; 72 C 90 sec.; 94 C 15 sec] 3 times    -   [94 C 15 sec.; 50 C 30 sec.; 72 C 90 sec] 35 times    -   72 C 10 min; 4 C hold        This band was subcloned into the EcoRV site of pBluescript II        and sequenced using the USB Sequence kit.

Screen of a Butterfly cDNA Library with Mosquito PCR Product. Using themosquito PCR product (SEQ ID NO:7) as a probe, a 3 day embryonic Preciscoenia gt10 cDNA library (generously provided by Sean Carroll) wasscreened. Filters were hybridized at 65° C. overnight in a solutioncontaining 5×SSC, 10% dextran sulfate, 5× Denhardt's, 200 μg/mlsonicated salmon sperm DNA, and 0.5% SDS. Filters were washed in0.1×SSC, 0.1% SDS at room temperature several times to removenonspecific hybridization. Of the 100,000 plaques initially screened, 2overlapping clones, L1 and L2, were isolated, which corresponded to theN terminus of butterfly ptc. Using L2 as a probe, the library filterswere rescreened and 3 additional clones (L5, L7, L8) were isolated whichencompassed the remainder of the ptc coding sequence. The full lengthsequence of butterfly ptc (SEQ ID NO:3) was determined by ABI automatedsequencing.

Screen of a Tribolium (beetle) Genomic Library with Mosquito PCR Productand 900 bp Fragment from the Butterfly Clone. A gem11 genomic libraryfrom Tribolium casteneum (gift of Rob Dennell) was probed with a mixtureof the mosquito PCR (SEQ ID NO:7) product and BstXI/EcoRI fragment ofL2. Filters were hybridized at 55 C overnight and washed as above. Ofthe 75,000 plaques screened, 14 clones were identified and the Saclfragment of T8 (SEQ ID NO:1), which crosshybridized with the mosquitoand butterfly probes, was subcloned into pBluescript.

PCR on Mouse cDNA Using Degenerate Primers Derived from RegionsConserved in the Four Insect Homologues. Two degenerate PCR primers(P4REV-(SEQ ID NO:16) GGACGAATTCYTNGANTGYTTYTGGGA-P22-(SEQ ID NO:17)CATACCAGCCAAGCTTGTCIGGCCARTGCAT) were designed based on a comparison ofptc amino acid sequences from fly (Drosophila melanogaster) (SEQ IDNO:6), mosquito (Anopheles gambiae) (SEQ ID NO:8), butterfly (Preciscoenia) (SEQ ID NO:4), and beetle (Tribolium casteneum) (SEQ ID NO:2). Irepresents inosine, which can form base pairs with all four nucleotides.P22 was used to reverse transcribe RNA from 12.5 dpc mouse limb bud(gift 0from David Kingsley) for 90 min at 37 C. PCR using P4REV (SEQ IDNO:17) and P22 (SEQ ID NO:18) was then performed on 1 l of the resultantcDNA under the following conditions:

-   -   94 C 4 min.; 72 C Add Taq;    -   [94 C 15 sec.-50 C 30 sec.-72 C 90 sec.] 35 times    -   72 C 10 min.-, 4 C hold        PCR products of the expected size were subcloned into the TA        vector (Invitrogen) and sequenced with the Sequenase Version 2.0        DNA Sequencing Kit (U.S. B.).

Using the cloned mouse PCR fragment as a probe, 300,000 plaques of amouse 8.5 dpc gt10 cDNA library (a gift from Brigid Hogan) were screenedat 65 C as above and washed in 2×SSC, 0.1% SDS at room temperature. 7clones were isolated, and three (M2, M4, and M8) were subcloned intopBluescript II. 200,000 plaques of this library were rescreened usingfirst, a 1.1 kb EcoRI fragment from M2 to identify 6 clones (M9-M16) andsecondly a mixed probe containing the most N terminal (Xhol fragmentfrom M2) and most C terminal sequences (BamHI/BgIII fragment from M9) toisolate 5 clones (M17-M21). M9, M10, M14, and M17-21 were subcloned intothe EcoRI site of pBluescript II (Strategene).

RNA Blots and In Situ Hybridizations in Whole and Sectioned MouseEmbryos:

Northerns. A mouse embryonic Northern blot and an adult multiple tissueNorthern blot (obtained from Clontech) were probed with a 900 bp EcoRlfragment from an N terminal coding region of mouse ptc. Hybridizationwas performed at 65° C. in 5×SSPE, 10× Denhardt's, 100 μg/ml sonicatedsalmon sperm DNA, and 2% SDS. After several short room temperaturewashes in 2×SSC, 0.05% SDS, the blots were washed at high stringency in0.1×SSC, 0.1% SDS at 50° C.

In situ hybridization of sections: 7.75, 8.5, 11.5, and 13.5 dpc mouseembryos were dissected in PBS and frozen in Tissue-Tek medium at −80° C.12-16 μm frozen sections were cut, collected onto VectaBond (VectorLaboratories) coated slides, and dried for 30-60 minutes at roomtemperature. After a 10 minute fixation in 4% paraformaldehyde in PBS,the slides were washed 3 times for 3 minutes in PBS, acetylated for 10minutes in 0.25% acetic anhydride in triethanolamine, and washed threemore times for 5 minutes in PBS. Prehybridization (50% formamide, 5×SSC,250 μg/ml yeast tRNA, 500 μg/ml sonicated salmon sperm DNA, and 5×Denhardt's) was carried out for 6 hours at room temperature in 50%formamide/5×SSC humidified chambers. The probe, which consisted of 1 kbfrom the N-terminus of ptc, was added at a concentration of 200-1000ng/ml into the same solution used for prehybridization, and thendenatured for five minutes at 80° C. Approximately 75 μl of probe wereadded to each slide and covered with Parafilm. The slides were incubatedovernight at 65° C. in the same humidified chamber used previously. Thefollowing day, the probe was washed successively in 5×SSC (5 minutes,65° C.), 0.2×SSC (1 hour, 65° C.), and 0.2×SSC (10 minutes, roomtemperature). After five minutes in buffer B1 (0.1M maleic acid, 0.15 MNaCl, pH 7.5), the slides were blocked for 1 hour at room temperature in1% blocking reagent (Boerhinger-Mannheim) in buffer B1, and thenincubated for 4 hours in buffer B1 containing the DIG-AP conjugatedantibody (Boerhinger-Mannheim) at a 1:5000 dilution. Excess antibody wasremoved during two 15 minute washes in buffer B1, followed by fiveminutes in buffer B3 (100 mM Tris, 100 mM NaCl, 5 nmM MgCl2, pH 9.5).The antibody was detected by adding an alkaline phosphatase substrate(350 μl 75 mg/ml X-phosphate in DMF, 450 μl 50 mg/ml NBT in 70% DMF in100 mls of buffer B3) and allowing the reaction to proceed overnight inthe dark. After a brief rinse in 10 mM Tris, 1 mM EDTA, pH 8.0, theslides were mounted with Aquamount (Lerner Laboratories).

Drosophila 5-transcriptional initiation region -gal constructs. A seriesof constructs were designed that link different regions of the ptcpromoter from Drosophila to a LacZ reporter gene in order to study thecis regulation of the ptc expression pattern. See FIG. 1. A 10.8 kbBamHI/BspMl fragment comprising the 5′-non-coding region of the mRNA atits 3′-terminus was obtained and truncated by restriction enzymedigestion as shown in FIG. 1. These expression cassettes were introducedinto Drosophila lines using a P-element vector (Thummel et al. (1988)Gene 74:445-456), which were injected into embryos, providing flieswhich could be grown to produce embryos. (See Spradling and Rubin (1982)Science 218:341-347 for a description of the procedure.) The vector useda pUC8 background into which was introduced the white gene to providefor yellow eyes, portions of the P-element for integration, and theconstructs were inserted into a polylinker upstream from the LacZ gene.The resulting embryos, larvae, and adults were stained using antibodiesto LacZ protein conjugated to HRP and the samples developed with OPD dyeto identify the expression of the LacZ gene. The staining pattern inembryos is described in FIG. 1, indicating whether there was stainingduring the early and late development of the embryo.

Isolation of a Mouse ptc Gene. Homologues of fly ptc (SEQ ID NO:6) wereisolated from three insects: mosquito, butterfly and beetle, usingeither PCR or low stringency library screens. PCR primers to six aminoacid stretches of ptc of low mutatability and degeneracy were designed.One primer pair, P2 and P4, amplified an homologous fragment of ptc frommosquito genomic DNA that corresponded to the first hydrophilic loop ofthe protein. The 345 bp PCR product (SEQ ID NO:7) was subcloned andsequenced and when aligned to fly ptc, showed 67% amino acid identity.

The cloned mosquito fragment was used to screen a butterfly gt 10 cDNAlibrary. Of 100,000 plaques screened, five overlapping clones wereisolated and used to obtain the full length coding sequence. Thebutterfly ptc homologue (SEQ ID NO:4) is 1,311 amino acids long andoverall has 50% amino acid identity (72% similarity) to fly ptc. Withthe exception of a divergent C-terminus, this homology is evenly spreadacross the coding sequence. The mosquito PCR clone (SEQ ID NO:7) and acorresponding fragment of butterfly cDNA were used to screen a beetlegemll genomic library. Of the plaques screened, 14 clones wereidentified. A fragment of one clone (T8), which hybridized with theoriginal probes, was subcloned and sequenced. This 3 kb piece containsan 89 amino acid exon (SEQ ID NO:2) which is 44% and 51% identical tothe corresponding regions of fly and butterfly ptc respectively.

Using an alignment of the four insect homologues in the firsthydrophilic loop of the ptc, two PCR primers were designed to a five andsix amino acid stretch which were identical and of low degeneracy. Theseprimers were used to isolate the mouse homologue using RT-PCR onembryonic limb bud RNA. An appropriately sized band was amplified andupon cloning and sequencing, it was found to encode a protein 65%identical to fly ptc. Using the cloned PCR product and subsequently,fragments of mouse ptc cDNA, a mouse embryonic cDNA library wasscreened. From about 300,000, plaques, 17 clones were identified and ofthese, 7 form overlapping cDNA's that comprise most of theprotein-coding sequence (SEQ ID NO:9).

Developmental and Tissue Distribution of Mouse ptc RNA. In both theembryonic and adult Northern blots, the ptc probe detects a single 8 kbmessage. Further exposure does not reveal any additional minor bands.Developmentally, ptc mRNA is present in low levels as early as 7 dpc andbecomes quite abundant by 11 and 15 dpc. While the gene is still presentat 17 dpc, the Northern blot indicates a clear decrease in the amount ofmessage at this stage. In the adult, ptc RNA is present in high amountsin the brain and lung, as well as in moderate amounts in the kidney andliver. Weak signals are detected in heart, spleen, skeletal muscle, andtestes.

In situ Hybridization of Mouse ptc in Whole and Section Embryos.Northern analysis indicates that ptc mRNA is present at 7 dpc, whilethere is no detectable signal in sections from 7.75 dpc embryos. Thisdiscrepancy is explained by the low level of transcription. In contrast,ptc is present at high levels along the neural axis of 8.5 dpc embryos.By 11.5 dpc, ptc can be detected in the developing lung buds and gut,consistent with its adult Northern profile. In addition the gene ispresent at high levels in the ventricular zone of the central nervoussystem, as well as in the zona limitans of the prosencephalon. ptc isalso strongly transcribed in the condensing cartilage of 11.5 and 13.5dpc limb buds, as well as in the ventral portion of the somites, aregion which is prospective sclerotome and eventually forms bone in thevertebral column. ptc is present in a wide range of tissues fromendodermal, mesodermal and ectodermal origin supporting its fundamentalrole in embryonic development.

Isolation of the Human ptc Gene. To isolate human ptc (hptc), 2×105plaques from a human lung cDNA library (HL3022a, Clonetech) werescreened with a 1 kbp mouse ptc fragment, M2-2. Filters were hybridizedovernight at reduced stringency (60° C. in 5×SSC, 10% dextran sulfate,5× Denhardt's, 0.2 mg/ml sonicated salmon sperm DNA, and 0.5% SDS). Twopositive plaques (H1 and H2) were isolated, the inserts cloned intopBluescript, and upon sequencing, both contained sequence highly similarto the mouse ptc homolog. To isolate the 5′ end, an additional 6×10⁵plaques were screened in duplicate with M2-3 EcoRI and M2-3 Xho I(containing 5′ untranslated sequence of mouse ptc) probes. Ten plaqueswere purified and of these, inserts were subcloned into pBluescript. Toobtain the full coding sequence, H2 was fully and H14, H20, and H21 werepartially sequenced. The 5.1 kbp of human ptc sequence (SEQ ID NO:18)contains an open reading frame of 1447 amino acids (SEQ ID NO: 19) thatis 96% identical and 98% similar to mouse ptc. The 5′ and 3′untranslated sequences of human ptc (SEQ ID NO:18) are also highlysimilar to mouse ptc (SEQ ID NO:19) suggesting conserved regulatorysequence.

Comparison of Mouse, Human, Fly and Butterfly Sequences. The deducedmouse ptc protein sequence (SEQ ID NO: 10) has about 38% identical aminoacids to fly ptc over about 1,200 amino acids. This amount ofconservation is dispersed through much of the protein excepting theC-terminal region. The mouse protein also has a 50 amino acid insertrelative to the fly protein. Based on the sequence conservation of ptcand the functional conservation of hedgehog between fly and mouse, oneconcludes that ptc functions similarly in the two organisms. Acomparison of the amino acid sequences of mouse (mptc) (SEQ ID NO:10),human (hptc) (SEQ ID NO: 19), butterfly (bptc) (SEQ ID NO:4) andDrosophila (ptc) (SEQ ID NO:6) is shown in the follwing table.

Alignment of Human, Mouse, Fly, and Butterfly ptc Homologs

HPTC MASAGNAAEPQDR--GGGGSGCIGAPGRPAGGGRRRRTGGLRRAAAPDRDYLHRPSYCDA MPTCMASAGNAA---------------GALGRQAGGGRRRRTGGPHRA-APDRDYLHRPSYCDA PTCM-----DRDSLPRVPDTHGD--VVDE---------KLFSDL---------YI-RTSWVDA BPTCMVAPDSEAPSNPRITAAHESPCATEA---------RHSADL---------YI-RTSWVDA*                                  .  ..          *. * *  ** HPTCAFALEQISKGKATGRKAPLWLRAKFQRLLFKLGCYIQKNCGKFLVVGLLIFGAFAVGLKA MPTCAFALEQISKGKATGRKAPLWLRAKFQRLLFKLGCYIQKNCGKFLVVGLLIFGAFAVGLKA PTCQVALDQIDKGKARGSRTAIYLRSVFQSHLETLGSSVQKHAGKVLFVAILVLSTFCVGLKS BPTCALALSELEKGNIEGGRTSLWIRAWLQEQLFILGCFLQGDAGKVLFVAILVLSTFCVGLKS  ** .. **.  * .... .*. .*  *  **   * . ** * *..*....* ****. HPTCANLETNVEELWVEVGGRVSRELNYTRQKIGEEAMFNPQLMIQTPKEEGANVLTTEALLQH MPTCANLETNVEELWVEVGGRVSRELNYTRQKIGEEAMFNPQLMIQTPKEEGANVLTTEALLQH PTCAQIHSKVHQLWIQEGGRLEAELAYTQKTIGEDESATHQLLIQTTHDPNASVLHPQALLAH BPTCAQIHTRVDQLWVQEGGRLEAELKYTAQALGEADSSTHQLVIQTAKDPDVSLLHPGALLEH*.... *..**.. ***.  ** ** . .**      **.***  .   ..*   *** * HPTCLDSALQASRVHVYMYNRQWKLEHLCYKSGELITET-GYMDQIIEYLYPCLIITPLDCFWE MPTCLDSALQASRVHVYMYNRQWKLEHLCYKSGELITET-GYMDQIIEYLYPCLIITPLDCFWE PTCLEVLVKATAVKVHLYDTEWGLRDMCNMPSTPSFEGIYYIEQILRHLIPCSIITPLDCFWE BPTCLKVVHAATRVTVHMYDIEWRLKDLCYSPSIPDFEGTHHIESIIDNVIPCAIITPLDCFWE*     *. * * .*. .* * ..*  ..    *    .. *. . ** *********** HPTCGAKLQSGTAYLLGKPPLR----WTNFDPLEFLEELK------KINYQVDSWEEMLNKAEV MPTCGAKLQSGTAYLLGKPPLR----WTNFDPLEFLEELK------KINYQVDSWEEMLNKAEV PTCGSQLL-GPESAVVIPGLNQRLLWTTLNPASVMQYMKQKMSEEKISFDFETVEQYMKRAAI BPTCGSKLL-GPDYPIYVPHLKHKLQWTHLNPLEVVEEVK-KL---KFQFPLSTIEAYMKRAGI *..* *     *  * *     ** ..*   .. .*      *. .   . *  ...*. HPTCGHGYMDRPCLNPADPDCPATAPNKNSTKPLDMALVLNGGCHGLSRKYMHWQEELIVGGTV MPTCGHGYMDRPCLNPADPDCPATAPNKNSTKPLDVALVLNGGCQGLSRKYMHWQEELIVGGTV PTCGSGYMEKPCLNPLNPNCPDTAPNKNSTQPPDVGAILSGGCYGYAAKHMHWPEELIVGGRK BPTCTSAYMKKPCLDPTDPHCPATAPNKKSGHIPDVAAELSHGCYGFAAAYMHWPEQLIVGGAT  .** .***.* .*.** ***** *    *..  *. ** * .   *** *.***** HPTCKNSTGKLVSAHALQTMFQLMTPKQMYEHFKGYEYVSHINWNEDKAAAILEAWQRTYVEVV MPTCKNATGKLVSAHALQTMFQLMTPKQMYEHFRGYDYVSHINWNEDRAAAILEAWQRTYVEVV PTCRNRSGHLRKAQALQSVVQLMTEKEMYDQWQDNYKVHHLGWTQEKAAEVLNAWQRNFSREV BFTCRNSTSALRSARALQTVVQLMGEREMYEYWADHYKVHQIGWNQEKAAAVLDAWQRKFAAEV.* .. *  *.***.. ***  ..**.   .   * .. * ...** .*.**** .   * HPTCHQSVAQNSTQK----VLSFTTTTLDDTLKSFSDVSVIRVASGYLLMLAYACLTMLRW-DC MPTCHQSVAPNSTQK----VLPFTTTTLDDILKSFSDVSVIRVASGYLLMLAYACLTMLRW-DC PTCEQLLRKQSRIATNYDIYVFSSAALDDILAKFSHPSALSIVIGVAVTVLYAFCTLLRWRDP BPTCRKI-TTSGSVSSAYSFYPFSTSTLNDILGKFSEVSLKNIILGYMFMLIYVAVTLIQWRDP .     .          *....*.***  **. *   .  *    . *   *...* * HPTCSKSQGAVGLAGVLLVALSVAAGLGLCSLIGISFNAATTQVLPFLALGVGVDDVFLLAHAF MPTCSKSQGAVGLAGVLLVALSVAAGLGLCSLIGISFNAATTQVLPFLALGVGVDDVFLLAHAF PTCVRGQSSVGVAGVLLMCFSTAAGLGLSALLGIVFNAASTQVVPFLALGLGVDHIFMLTAAY BPTCIRSQAGVGIAGVLLLSITVAAGLGFCALLGIPFNASSTQIVFFLALGLGVQDMFLLTHTY HPTCSETGQNKRIPFEDRTGECLKRTGASVALTSISNVTAFFMAALIPIPALRAFSLQAAVVVV MPTCSETGQNKRIPFEDRTGECLKRTGASVALTSISNVTAFFMAALIPIPALRAFSLQAAVVVV PTCAESN------RREQTKLILKKVGFSILFSACSTAGSFFAAAFIPVPALKVFCLQAAIVMC BPTCVEQAGD--VPREERTGLVLKKSGLSVLLASLCNVMAFLAAALLPIPAFRVFCLQAAILLL HPTCFNFAMVLLIFPAILSMDLYRREDRRLDIFCCFTSPCVSRVIQVEFQAYTDTHDNTRYSPP MPTCFNFAMVLLIFPAILSMDLYRPEDRRLDIFCCFTSPCVSRVIQVEPQAYTEPHSNTRYSPP PTCSNLAAALLVFPAMISLDLRRRTAGRADIFCCCF-PVWKEQPKVAPPVLPLNNNNGR---- BPTCFNLGSILLVFPAMISLDLRRRSAAPADLLCCLM-P---ESP------LPKKKIPER---- HPTCPPYSSHSFAHETQITMQSTVQLRTEYDPHTHVYYTTAEPRSEISVQPVTVTQDT LSCQSP MPTCPPYTSHSFAHETHITMQSTVQLRTEYDPHTHVYYTTAEFRSEISVQPVTVTQDNLSCQSP PTC --------------------------------- GARHPKSCNNNRVPLPAQNPLLEQFA BPTC----------------------------------- AKTRKNDKTHRID-TTRQPLDPDVS HPTCESTSSTRDLLSQFSDSSLHCLEPPCTKWTLSSFAEKHYAPFLLKPKAKVVVIFLFLGLLG MPTCESTSSTRDLLSQFSDSSLHCLEPPCTKWTLSSFAEKHYAPFLLKPKAKVVVILLFLGLLG PTCDIPGSS------------HSLASF----SLATFAFQHYTPFLMRSWVKFLTVMGFLAALI BPTCENVTKT------------CCL-SV----SLTKWAKNQYAPFIMRPAVKVTSMLALIAVIL HPTCVSLYGTTRVRDGLDLTDIVPRETREYDFIAAQFKYFSFYNMYIVTQKA-DYPNIQHLLYD MPTCVSLYGTTRVRDGLDLTDIVPRETREYDFIAAQFKYFSFYNMYIVTQKA-DYPNIQHLLYD PTCSSLYASTRLQDGLDIIDLVPKDSNEHKFLDAQTRLFGFYSMYAVTQGNFEYPTQQQLLRD BPTCTSVWGATKVKDGLDLTDIVPENTDEHEFLSRQEKYFGFYNMYAVTQGNFEYPTNQKLLYE HPTCLHRSFSNVKYVMLEENKQLPKMWLHYFRDWLQGLQDAFDSDWETGKIMPNN-YKNGSDDG MPTCLHKSFSNVKYVMLEENKQLPQMWLHYFRDWLQGLQDAFDSDWETGRIMPNN-YKNGSDDG PTCYHDSFVRVPHVIKNDNGGLPDFWLLLFSEWLGNLQKIFDEEYRDGRLTKECWFPNASSDA BPTCYHDQFVRIPNIIKNDNGGLTKFWLSLFRDWLLDLQVAFDKEVASGCITQEYWCKNASDEG HPTCVLAYKLLVQTGSRDKPIDISQLTK-QRLVDADGIINPSAFYIYLTAWVSNDPVAYAASQA MPTCVLAYKLLVQTGSRDKPIDISQLTK-QRLVDADGIINPSAFYIYLTAWVSNDPVAYAASQA PTCILAYKLIVQTGHVDNPVDKELVLT-NRLVNSDGIINQRAFYNYLSAWATNDVFAYGASQG BPTCILAYKLMVQTGHVDNPIDKSLITAGHRLVDKDGIINPKAFYNYLSAWATNDALAYGASQG HPTCNIRPHRPEWVHDKADYMPETRLRIPAAEPIEYAQFPFYLNGLRDTSDFVEAIEKVRTICS MPTCNIRPHRPEWVHDKADYMPETRLRIPAAEPIEYAQFPFYLNGLRDTSDFVEAIEKVRVICN PTCKLYPEPRQYFHQPNEY----DLKIPKSLPLVYAQMPFYLHGLTDTSQIKTLIGHIRDLSV BPTCNLKPQPQRWIHSPEDV----HLEIKKSSPLIYTQLPFYLSGLSDTDSIKTLIRSVRDLCL HPTCNYTSLGLSSYPNGYPFLFWEQYIGLPHWLLLFISVVLACTFLVCAVFLLNPWTAGIIVMV MPTCNYTSLGLSSYPNGYPFLFWEQYISLRHWLLLSISVVLACTFLVCAVFLLNPWTAGIIVMV PTCKYEGFGLPNYPSGIPFIFWEQYMTLRSSLAMILACVLLAALVLVSLLLLSVWAAVLVILS BPTCKYEAKGLPNFPSGIPFLFWEQYLYLRTSLLLALACALGAVFIAVMVLLLNAWAAVLVTLA HPTCLALMTVELFGMMGLIGIKLSAVPVVILIASVGIGVEFTVHVALAFLTAIGDKNRRAVLAL MPTCLALMTVELFGMMGLIGIKLSAVPVVILIASVGIGVEFTVHVALAFLTAIGDKNHRAMLAL PTCVLASLAQIFGAMTLLGIKLSAIPAVILILSVGMMLCFNVLISLGFMTSVGNRQRRVQLSM BPTCLATLVLQLLGVMALLGVKLSAMPPVLLVLAIGRGVHFTVHLCLGFVTSIGCKRRRASLAL HPTCEHMFAPVLDGAVSTLLGVLMLAGSEFDFIVRYFFAVLAILTILGVLNGLVLLPVLLSFFG MPTCEHMFAPVLDGAVSTLLGVLMLAGSEFDFIVRYFFAVLAILTVLGVLNGLVLLPVLLSFFG PTCQMSLGPLVHGMLTSGVAVFMLSTSPFEFVIPHFCWLLLVVLCVGACNSLLVFPILLSMVG BPTCESVLAPVVHGALAAALAASMLA.ASEFGFVARLFLRLLLALVFLGLIDGLLFFPIVLSILO HPTCPYPEVSPANGLNRLPTPSPEPPPSVVRFAMPPGHTHSGSDSSDSEYSSQTTVSGLSE-EL MPTCPCPEVSPANGLNRLPTPSPEPPPSVVRFAVPPGHTNNGSDSSDSEYSSQTTVSGISE-EL PTCPEAELVPLEHPDRISTPSPLPVRSSKRSGKSYVVQGSRSSRGSCQKSHHHHHKDLNDPSL BPTCPAAEVRPIEHPERLSTPSPKCSPIHPRKSSSSSGGGDKSSRTS--KSAPRPC----APSL HPTCRHYEAQQGAGGPAHQVIVEATENPVFAHSTVVHPESRHHPPSNPRQQPHLDSGSLPPGRQ MPTCRQYEAQQGAGGPAHQVIVEATENPVFARSTVVHPDSPHQPPLTPRQQPHLDSGSLSPGRQ PTCTTITEEPQSWKSSNSSIQMPNDWTYQPREQ--RPASYAAPPPAYHKAAAQQHHQHQGPPT BPTCTTITEEPSSWHSSAHSVQSSMQSIVVQPEVVVETTTYNGSDSASGRSTPTKSSHGGAITT HPTCGQQPRRDPPREGLWPPLYRPRRDAFEISTEGHSGPSNRARWGPRGARSHNPPNPASTAMG MPTCGQQPRRDPPREGLRPPPYRPRRDAFEISTEGHSGPSNRDRSGPRGARSHNPRNPTSTAMG PTCTPPPPFPTA-----------------YPPELQSIVVQPEVTVETTHS-----------DS BPTCTKVTATANIKVEVVTPSDRKSRRSYHYYDRRRDRDEDRDRDRERDRDRDRDRDRDRDRDR HPTCSSVPGYCQPITTVTASASVTVAVHPPPVPGPGRNPRGGLCPGY---PETDHGLFEDPHVP MPTCSSVPSYCQPITTVTASASVTVAVHPP--PGPGRNPRGGPCPGYESYPETDHGVFEDPHVP PTCNT--------TKVTATANIKVELAMP-----GPAVRS---YNFTS--------------- BPTCDR--------DRERSRERDRP.DRYRD-----EPDHPA---SPRENGRDSGHE------- HPTCFHVRCERRDSKVEVIELQDVECEERPRGSSSN MPTC FHVRCERRDSKVEVIELQDVECEERPWGSSSNPTC -------------------------------- BPTC --------------------------SDSSRH

The identity of ten other clones recovered from the mouse library is notdetermined. These cDNAs cross-hybridize with mouse ptc sequence, whilediffering as to their restriction maps. These genes encode a family ofproteins related to the patched protein. Alignment of the human andmouse nucleotide sequences, which includes coding and noncodingsequence, reveals 89% identity.

Radiation hybrid mapping of the human ptc gene. Oligonucleotide primersand conditions for specifically amplifying a portion of the human ptcgene from genomic DNA by the polymerase chain reaction were developed.This marker was designated STS SHGC-8725. It generates an amplificationproduct of 196 bp, which is observed by agarose gel electrophoresis wheno human DNA is used as a template, but not when rodent DNA is used.Samples were scored in duplicate for the presence or absence of the 196bp product in 83 radiation hybrid DNA samples from the Stanford G3Radiation Hybrid Panel (purchased from Research Genetics, Inc.) Bycomparison of the pattern of G3 panel scores for those with a series ofGenethon meiotic linkage 5 markers, it was determined that the human ptcgene had a two point lod score of 1,000 with the meiotic marker D9S287,based on no radiation breaks being observed between the gene and themarker in 83 hybrid cell lines. These results indicate that the ptc genelies within 50-100 kb of the marker. Subsequent physical mapping in YACand BAC clones confirmed this close linkage estimate. Detailed mapinformation can be obtained from http://www.shgc.stanford.edu.

Analysis of BCNS mutations. The basal cell nevus syndrome has beenmapped to the same region of chromosome 9q as was found for ptc. Aninitial screen of EcoRI digested DNA from probands of 84 BCNS kindredsdid not reveal major rearrangements of the ptc gene, and so screeningwas performed for more subtle sequence abnormalities. Using vectorettePCR, by the method according to Riley et al. (1990) N.A.R. 18:2887-2890,on a BAC that contains genomic DNA for the entire coding region of ptc,the intronic sequence flanking 20 of the 24 exons was determined. Singlestrand conformational polymorphism analysis of PCR-amplified DNA fromnormal individuals, BCNS o patients and sporadic basal cell carcinomas(BCC) was performed for 20 exons of ptc coding sequence. The amplifiedsamples giving abnormal bands on SSCP were then sequenced.

In blood cell DNA from BCNS individuals, four independent sequencechanges were found; two in exon 15 and two in exon 10. One 49 year oldman was found to have a sequence change in exon 15. His affected sisterand daughter have the same alteration, but three unafflicted relativesdo not. His blood cell DNA has an insertion of 9 base pairs atnucleotide 2445 of the coding sequence, resulting in the insertion ofthree amino acids (PNI) after amino acid 815. Because the normalsequence preceding the insertion is also PNI, a direct repeat has beenformed.

The second case of an exon 15 change is an 18 year old woman whodeveloped jaw cysts at age 9 and BCCs at age 6. The developmentaleffects together with the BCCs indicate that she has BCNS, although noneof her relatives are known to have the syndrome. Her blood cell DNA hasa deletion of 11 bp, removing the sequence ATATCCAGCAC (SEQ ID NO: 20)at nucleotides 2441 to 2452 of the coding sequence. In addition,nucleotide 2452 is changed from a T to an A. The deletion results in aframeshift that is predicted to truncate the protein after amino acid813 with the addition of 9 amino acids. The predicted mutant protein istruncated after the seventh transmembrane domain. In Drosophila, a ptcprotein that is truncated after the sixth transmembrane domain isinactive when ectopically expressed, in contrast to the full-lengthprotein, suggesting that the human protein is inactivated by the exon 15sequence change. The patient with this mutation is the first affectedfamily member, since her parents, age 48 and 50, have neither BCCs norother signs of the BCNS. DNA from both parents' genes have the normalnucleotide sequence for exon 15, indicating that the alteration in exon15 arose in the same generation as did the BCNS phenotype. Hence herdisease is the result of a new mutation. This sequence change is notdetected in 84 control chromosomes.

Analysis of sporadic basal cell carcinomas. To determine whether ptc isalso involved in BCCs that are not associated with the BCNS or germlinechanges, DNA was examined from 12 sporadic BCCS. Three alterations werefound in these tumors. In one tumor, a C to T transition in exon 3 atnucleotide 523 of the coding sequence changes a highly conserved leucineto phenylalanine at residue '175 in the first putative extracellularloop domain Blood cell DNA from the same individual does not have thealteration, suggesting that it arose somatically in the tumor. SSCP wasused to examine exon 3 DNA from, 60 individuals who do not have BCNS,and found no changes from the normal sequence. Two other sporadic BCCshave deletions o encompassing exon 9 but not extending to exon 8.

The existence of sporadic and hereditary forms of BCCs is reminiscent ofthe characteristics of the two forms of retinoblastoma. This parallel,and the frequent deletion in tumors of the copy of chromosome 9qpredicted by linkage to carry the wild-type allele, demonstrates thatthe human ptc is a tumor suppressor gene. ptc represses a variety ofgenes, including growth factors, during Drosophila development and mayhave the same effect in human skin. The often reported large body sizeof BCNS patients also could be due to reduced ptc function, perhaps dueto loss of control of growth factors. The C to T transition identifiedin ptc in the sporadic BCC is also a common genetic change in the p53gene in BCC and is consistent with the role of sunlight in causing thesetumors. By contrast, the inherited deletion and insertion mutationsidentified in BCNS patients, as expected, are not those characteristicof ultraviolet mutagenesis.

The identification of the ptc mutations as a cause of BCNS links a largebody of developmental genetic information to this important humandisease. In embryos lacking ptc function part of each body segment istransformed into an anterior-posterior mirror-image duplication ofanother part. The patterning changes in ptc mutants are due in part toderepression of another segment polarity gene, wingless, a homolog ofthe vertebrate Wnt genes that encodes secreted signaling proteins. Innormal embryonic development, ptc repression of wg is relieved by the Hhsignaling protein, which emanates from adjacent cells in the posteriorpart of each segment. The resulting localized wg expression in eachsegment primordium organizes the pattern of bristles on the surface ofthe animal. The ptc gene inactivates its own transcription, while Hhsignaling induces ptc transcription.

In flies two other proteins work together with Hh to activate targetgenes: the ser/thr kinase fused and the zinc finger protein encoded bycubitus interruptus. Negative regulators working together with ptc torepress targets are protein kinase A and costal2. Thus, mutations thatinactivate human versions of protein kinase A or costal2, or that causeexcessive activity of human hh, gli, or a fused homolog, may modify theBCNS phenotype and be important in tumorigenesis.

In accordance with the subject invention, mammalian patched genes,including the mouse and human genes, are provided, which can serve manypurposes. Mutations in the gene are found in patients with basal cellnevus syndrome, and in sporadic basal cell carcinomas. The autosomaldominant inheritance of BCNS indicates that patched is a tumorsuppressor gene. The patched protein may be used in a screening foragonists and antagonists, and for assaying for the transcription of ptcmRNA. The protein or fragments thereof may be used to produce antibodiesspecific for the protein or specific epitopes of the protein. Inaddition, the gene may be employed for investigating embryonicdevelopment, by screening fetal tissue, preparing transgenic animals toserve as models, and the like.

As described above, patients with basal cell nevus syndrome have a highincidence of multiple basal cell carcinomas, medulloblastomas, andmeningiomas. Because somatic ptc mutations have been found in sporadicbasal cell carcinomas, we have screened for ptc mutations in severaltypes of sporadic extracutaneous tumors. We found that 2 of 14 sporadicmedulloblastomas bear somatic nonsense mutations in one copy of the geneand also deletion of the other copy. In addition, we identifiedmis-sense mutations in ptc in two of seven breast carcinomas, one ofnine meningiomas, and one colon cancer cell line. No ptc gene mutationswere detected in 10 primary colon carcinomas and eighteen bladdercarcinomas.

BCNS³ (OMIM #109400) is a rare autosomal dominant disease with diversephenotypic abnormalities, both tumorous (BCCs, medulloblastomas, andmeningiomas) and developmental (misshapen ribs, spina bifida occults,and skull abnormalities; Gorlin, R. J. (1987) Medicine 66:98-113). TheBCNS gene was mapped to chromosome 9q22.3 by linkage analysis of BCNSfamilies and by LOH analysis in sporadic BCCs (Gallani, M. R. et al.(1992) Cell 69:111-117). LOH in sporadic medulloblastomas has beenreported in the same chromosome region (Schofield, D. et al. (1995) Am JPathol 146:472-480). Recently, the human homologue of the Drosophilapatched (PTCII) gene has been mapped to the BCNS region (Hahn, H. et al.(1996) Cell 85:841-851; Johnson, R. L. et al. (1996) Science272:1668-1671; Gallani, M. R. et al (1996) Nat Genet 14:78-81; Xie, J.et al. (1997) Genes Chromosomes Cancer 18:305-309), and mutations inthis gene have been found in the blood DNA of BCNS patients and in theDNA of sporadic BCCs (Hahn, H. et al., supra; Johnson, R. L. et al.,supra; Gallani, M. R. et al., supra; and Chidambaram, A. et al. (1996)Cancer Res 36:4599-4601). ptc appears to function as a tumor suppressorgene; inactivation abrogates its normal inhibition of the hedgehogsignaling pathway. Because of the wide variety of tumors in patents withthe BCNS and wide tissue distribution of ptc gene expression, we havebegun screening for ptc gene mutations in several types of humancancers, especially those present in increased numbers in BCNS patients(medulloblastomas), those in tissues derived embryologically fromepidermis (breast carcinomas) and those with chromosome 9q LOG (bladdercarcinomas; see Cairns, P. et al. (1993) Cancer Res 53:1230-1232; andSidransky, D. et al. (1997) NEJM 326:737-740).

To further study the roles of ptc in development and in tumorigenesis,we have constructed mice lacking ptc function. By homologousrecombination, part of ptc exon 1 (including the putative start codon)and all of exon 2 were replaced with lacZ and a neomycin resistance gene(FIG. 3) (DNA from the ptc genomic locus was isolated from a 129SVgenomic phage library [Stratagene]. Exons 1-15 of human PTC (1) weremapped by PCR and sequencing. The 3′ arm of homology was a 3.5 kbEcoRI-BamHI fragment from the second intron that gained a BamHI sitefrom pBSII [Stratagene] and was cloned into the BamHI site of pPNT[Tybulewicz, et al. (1991) Cell 65:1153]. A cassette containing the genefor nuclear localized b-galactosidase, followed by the mP1 intron andpolyA tail was excised from pNLacF [Mercer, et al. (1991) Neuron 7:703]and cloned into the Xho I site of pPNT using Xho I and Sal I linkers.The 5′ arm of homology was a 6.5 kb Xho I to Nru I fragment that wascloned into the Xho I site upstream of lacZ via a Sal I linker. The NruI site is in the first ptc exon. The resulting plasmid, KO1, waslinearized with Xho I and electroporated into RI ES cells that weresubjected to double selection and analyzed by Southern blot [Joyner, A.L. Gene Targeting: A Practical Approach. Oxford University Press, NewYork, 1993, pp.33-61]. Targeted clones were expanded and used forinjection into C57B1/6 blastocysts [Hogan, B. et al. Manipulating theMouse Embryo: A Laboratory Manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor, 1994, pp.196-204]. Protein made from any alternativeATG would lack the first proposed transmembrane domain, flipping theorientation of the protein in the membrane. Three independent ES cloneswere used to make chimeras that were bred to B6D2F1 animals to generateheterozygous mice on a mixed background. Interbreeding of heterozygotesproduced no homozygous animals among 202 offspring examined. Analysis ofembryos from timed matings suggested that ptc^(−/−) embryos die betweenembryonic day (E) 9.0 and E10.5, with the first gross phenotypesappearing by E8. In ptc^(−/−) embryos, the neural tube failed to closecompletely and was overgrown in the head folds, hindbrain and spinalcord (FIG. 4, A to C). Embryonic lethality may have been due to abnormaldevelopment of the heart (FIG. 4B), which never beats.

In flies Ptc protein inhibits ptc transcription. By inhibiting Ptcfunction, Hh increases production of Ptc which may then bind availableHh and limit the range or duration of effective Hh signal (Y. Chen andG. Struhl, (1996) Cell 87:553). Hh signaling also post-transcriptionallyregulates the zinc finger protein cubitus interruptus (ci) (C. K. Motznyand R. Holmgren, (1996) Mech Dev 52:137; Domínguez, et al. (1996)Science 272:1621; Hepker, et al. (1997) Development 124:549; Aza-Blanc,et al., (1997) Cell 89:1043). In vertebrates, Sonic hedgehog (Shh)signaling induces transcription of both ptc and a ci homolog, Gli(Goodrich, et al. (1996) Genes Devel. 10:301; Marigo, et al. (1996)Development 122:1225; Concordet, et al., (1996) Development 122:2835;Marigo, et al (1996) Dev. Biol. 180:273). Derepression of ptc and Gli inptc^(−/−) mice should therefore reveal where Ptc is normally active.

ptc and Gli expression was greatly increased in ptc^(−/−) embryos. Inptc^(+/−) mice expression of the lacZ gene fused to the first ptc exonduring targeting accurately reported the pattern of ptc transcription(FIG. 4, C and D). In ptc^(−/−) embryos expression of ptc-lacZ wasextensively derepressed starting at about E8.0 in the anterior neuraltube and spreading posteriorly by E8.75 (FIG. 4, C and E). Derepressionwas germ layer-specific: both ptc-lacZ and Gli were expressed throughoutthe ectoderm and mesoderm, but not in the endoderm (FIG. 4, D to G). ptcexpression may be excluded from the endoderm in order to avoidinterfering with Shh signaling from the endoderm to the mesoderm(Roberts et al., (1995) Development 121:3163). A differentialrequirement for Ptc may distinguish the germ layers.

As revealed by ptc mutants, an early site of Ptc activity is the neuraltube, where Shh and Ptc act antagonistically to determine cell fates.Shh induces the floor plate and motor neurons in the ventral neural tube(Echelard et al., (1993) Cell 75:1417; Roelink et al., (1994) Cell76:761; Roelink et al., (1995) Cell 81:445-455). These cell types failto form in Shh mutants (Chiang et al., (1996) Nature 383:407). Highlevels of Shh produced by the notochord may induce floor plate bycompletely inactivating Ptc (Echelard et al., (1993) supra; Roelink etal., (1994) supra; Roelink et al., (1995) supra). If so, elimination ofptc function might cause floor plate differentiation throughout theneural tube. Prospective floor plate cells transcribe the forkheadtranscription factor HNF3b first and then Shh itself (Echelard et al.,(1993) supra; Roelink et al., (1994) supra; Roelink et al., (1995)supra). In E8.5 ptc mutants, transcription of HNF3b and Shh was expandeddorsally (FIG. 5, A to C). Ectopic Shh expression was most extensive inthe anterior, where transcripts could be detected throughout theneurepithelium (FIG. 5, B and C). Cells in this region were in a singlelayer with basal nuclei, like floor plate cells that are normallyrestricted to the ventral midline (FIG. 5, D and E). Expression of theintermediate neural tube marker Pax6 (C. Walther and P. Gruss, (1991)Development 113:1435) was completely absent from ptc mutant embryos,suggesting that only ventral, and not ventrolateral, cell fates arespecified (FIG. 5, F and G).

Dorsalizing signals from the surface ectoderm (Dickinson, et al. (1995)Development 121:2099; Liem, et al. (1995) Cell 82:969) could conferdorsal cell fates even in the absence of ptc function. In E8-E9 ptchomozygotes the dorsal neural tube marker Pax3 was not expressed in theanterior neural tube, but was transcribed in a very small region at thedorsal-most edge of the posterior neural tube (FIG. 5, H to J). Inaddition erb-b3 transcription, which marks migratory neural crest cells(FIG. 5K) (H. U. Wang and D. J. Anderson, (1997) Neuron 18:383), was notdetected in the somites of ptc mutants (FIG. 5L). We conclude that onlylimited dorsal fate determination occurs in the absence of ptc. BMPsignals maintain dorsal gene expression (Dickinson, et al. (1995) supra;Liem, et al. (1995) supra), so either ptc is required for BMPs to workor BMP signaling is ineffective in most cells expressing Shh targets.

Ventralization of the neural tube in ptc mutants occurred withoutaffecting cell identity along the rostrocaudal axis. In ptc^(−/−)embryos, cells in the anterior neural tube expressed the forebrainmarker Nkx2.1 (Shimamura, et al. (1995) Development 121:3923) and cellsin the spinal cord transcribed low levels of hoxbl (Wilkinson, et al.(1989) Nature 341:405) (FIG. 5, M and N). hoxbl was not transcribed inthe fourth rhombomere of ptc mutants (FIG. 5, N). This may reflect atransformation of hindbrain cells to floor plate, since hoxbl isexcluded from the midline of wild-type embryos. Conversely, in theanterior, Nkx2.1 expression was expanded dorsally in mutants compared towild-type embryos (FIG. 5, M).

ptc^(+/−) mice had phenotypes similar to those of BCNS patients: theywere larger than their wild-type littermates [30.72±3.83 (average±SD;n=29) vs. 26.54±2.51 (n=39) at 2-3 months; P=0.000001], a small fraction(3 of 389 mice examined) had hindlimb defects such as extra digits orsyndactyly (FIG. 6A) or obvious soft tissue tumors (1 of 243) and manydeveloped brain tumors (see below).

Of 243 ptc^(+/−) mice which were between the ages of 2 and 9 months andwere not sacrificed for other studies, 18 died or were euthanizedbecause of sickness. No wild-type littermates died. Ten of the affectedheterozygotes were autopsied and eight were found to have large growthsin the cerebellum that resembled medulloblastomas (FIG. 6, B and C).Human medulloblastomas are believed to arise from a “primitiveneurectodermal” cell type (J. P. Provias and L. E. Becker, (1996) JNeurooncol 29:35). They are most common in children, can be metastaticor non-metastatic, and can have glial and neuronal properties. Thehistology of tumors from ptc^(+/−) mice was similar to that of humanmedulloblastoma: tumor cells were small, with dark carrot-shaped nucleiand little cytoplasm (FIG. 6, D and E), and although a subset expressedneurofilament protein and synaptophysin (FIG. 6F) (For immunostaining,two tumors were fixed and embedded in paraffin. Tissue sections (4 mm)were cleared and dehydrated, treated with 3% hydrogen peroxide and thenwith a dilution of 1:10 normal rabbit serum (Vector Laboratories).Anti-synaptophysin (Boehringer-Mannheim) was used at a dilution of 1:5and anti-neurofilament protein (Dako) at 1:50. Antibody binding wasvisualized with a peroxidase Vectastain Elite ABC kit (VectorLaboratories). Nuclei were counterstained with hematoxylin. Likeanti-synaptophysin, anti-neurofilament staining appeared in processes ofthe tumor cells.), the majority of cells appeared undifferentiated. Ofthe two autopsied animals without apparent medulloblastomas, one had alarge tumor growing out of its rib muscle and the other died for unknownreasons. Medulloblastomas and soft tissue tumors were also observed inptc^(+/−) mice maintained on an inbred 129SV background: 6 of 27 hadobvious medulloblastomas; 2 of 27 had tumors in the muscle of their leg;and 3 of 27 died but were not examined.

The ptc and Gli genes were strongly transcribed in the brain tumors butnot in surrounding tissue (FIG. 7, A and B; n=3 of 3 tumors examined).There was no detectable increase in Shh expression (FIG. 7C). To assessthe incidence of medulloblastomas, brains from 47 asymptomatic ptc^(+/−)mice were randomly collected and stained with X-gal. Nine brainscontained medulloblastomas that were easily recognized by theirdisorganized morphology and intense ptc-lacZ expression (FIG. 7D).Medulloblastomas were observed in 7 of 23 (30.4%) ptc^(+/−) mice at 12to 25 weeks of age, 1 of 12 (8.3%) mice at 9 to 10 weeks and 1 of 12(8.3%) mice at 5 weeks. Tumors can therefore arise as early as 5 weekspostnatally, but they increase in severity and frequency as the animalages.

We looked for changes in ptc-lacZ expression that might reflect earlystages of tumorigenesis. At all stages examined, about half of theanimals [50% at 5 to 10 weeks (n=24), 56.5% at 12 to 25 weeks (n=23)]exhibited regions of increased X-gal staining on the surface of thecerebellum (FIG. 7E). These regions were usually lateral and oftenextended down into the fissures separating the folia (FIG. 7, E and F).The mouse medulloblastomas may arise from these cells, which aresuperficial to the molecular layer of the cerebellum (FIG. 7F). Duringfetal development, prospective cerebellar granule cells proliferate inthe external granule layer (EGL), the outermost layer of the cerebellum.Granule cells then leave and migrate past the Purkinje cells to form theinternal granule cell layer of the adult animal, gradually depleting theEGL. The remnants of the fetal EGL have been proposed to be a source ofhuman medulloblastoma progenitors, a hypothesis consistent with thehigher frequency of these tumors in children (L. Stevenson and F.Echlin, (1934) Arch. Neurol. Psychiat. 31:93; Kadin, et al. (1970) JNeuropathol Exp Neurol 29:583).

The abundance of cerebellar ptc transcripts was reduced by about 50% inthe ptc^(+/−) mice compared to wild-type littermates (FIG. 7G). Thisreduction could lead to ectopic expression of Shh target genes and touncontrolled cell proliferation. Brain tumors might arise from Ptchaploinsufficiency alone, from additional mutations in the second ptcallele, or from a combination of ptc mutations with mutations in othertumor suppressor loci. We have not observed BCCs in ptc^(+/−) mice,perhaps because somatic inactivation of the second ptc gene is requiredas it is in human BCCs.

Our analysis has revealed that Ptc controls growth and pattern formationin early neural development and in the adult cerebellum. Autoregulationof ptc occurs in vertebrates as it does in flies, and the balancebetween Hh and Ptc activities appears critical for normal development.The importance of Ptc dosage is emphasized by the phenotype of theptc^(+/−) mice, which develop a tumor type observed in the correspondinghuman cancer predisposition syndrome. Medulloblastoma is a commonchildhood brain tumor and the prognosis remains grim. The Hh/Ptc pathwaymay provide new diagnostic tools and new insights into tumorigenesisthat may be directed toward potential therapies.

Materials and Methods

Clinical Materials. Diagnoses of all tumors were confirmedhistologically. Cell lines were obtained from the America Type CultureCollection. DNA was extracted from tumors or matched normal tissue(peripheral blood leukocytes or skin) as described (Cogen, P. H. et al.(1990) Genomics 8:279-285; and Sambrook, J. et al. Molecular Cloning: ALaboratory Manual, Ed. 2, Vol. 2, pp. 9.17-9.19, Cold Spring Harbor,N.Y. (1989)).

PCR and Heteroduplex Analysis. PCR amplification and heteroduplex/SSCPanalysis were performed as described (Johnson, R. L. et al., supra;Spritz, R. A. et al. (1992) Am J Hum Genet 51:1058-1065). Primers usedand intron/exon boundary sequences of the ptc gene were derived asreported previously (Johnson, R. L. et al., supra) and are shown inTable 1. Primers for exon 1 and 2 were from Hahn et al (supra).

Sequence Analysis. Exon segments exhibiting bands were reamplified andwere sequenced directly using the Sequenase sequencing kit according tothe protocol recommended by the manufacturer (United States BiochemicalCorp.). A second sequencing was performed using independently amplifiedPCR products to confirm the sequence change. The amplified PCR productsfrom each tumor were also cloned into the plasmid vector pCR 2.1(InVitrogen), followed by sequence analysis of at least four independentclones. The sequence alteration was confirmed from at least twoindependent clones. Simplified amplification of specific allele analysiswas performed according to Lei and Hall (Lei, X. and Hall, B. G. (1994)Biotechniques 16:44-45).

Allele Loss Analysis. Microsatellites used for allelic loss analysiswere D9S109, DpS119, D9S127, and D9S287 described in the CHLC humanscreening set (Research Genetics). A part of the ptc intron I sequencewas tested for polymorphism in a control population and found to bepolymorphic in 80% of the samples tested. This microsatellite was usedfor analysis of ptc gene allelic loss in bladder carcinomas. The primersequences are as follows: forward primer, 5′-CTGAGCAGATTTCCCAGGTC-3′(SEQ ID NO: 21); and reverse primer, 5′-CCTCAGACAGACCTTTCCTC-3′ (SEQ IDNO: 22). The PCR cycling for this newly isolated marker was 4 mm. at 95°C., followed by 30 cycles of 40s at 95° C. 2 min. at 60° C., and 1 min.at 72° C. PCR products were separated on 6% polyacrylamide gels andexposed to film.

Results and Discussion

Intronic boundaries were determined for 22 exons of ptc by sequencingvectorette PCR products derived from BAC 192J22 (Johnson R. L., supra;Table 1). Our findings are in agreement with those of Hahn et al.(supra), expect that we find exon 12 is composed of 2 separate exons of126 and 119 nucleotides. This indicates that ptc is composed of 23coding exons instead of 22. In addition, we find that exons 3, 4, 10,11, 17, 21, and 23 differ slightly in size than reported previously(Hahn et al., supra). Of 63 tumors studied, 14 were sporadicmedulloblastomas, and 9 were sporadic meningiomas. These 23 tumors wereexamined for allelic deletions by genotyping of tumor and blood DNA withmicrosatellite markers that flank the ptc gene: D9S119, D9S196, D9S287,D9S127, and D9S109. Four of 14 medulloblastomas had LOH. Two of themedulloblastomas, both of which had LOH, had mutations (med34 and med36;see Cogen, P. H. et al., supra), which are predicted to result intruncated proteins (Table 2). DNA samples from the blood of thesepatients lack these mutations, indicating that they both are somaticmutations. med34 also has allelic loss on 17p (Cogen, P. H. et al.,supra). We were unable to detect ptc gene mutations by heteroduplexanalysis in the other two medulloblastomas bearing LOH on 9q. Thepathological features of these two tumors differed in that med34 belongsto the desmoplastic subtype, whereas med36 is of the classic type,indicating that ptc mutations in medulloblastomas are not restricted toa specific subtype.

TABLE 1 Primers and boundary sequences of PTCH Reading Exon5′ Boundary^(a) Nucleotide Position^(b) Exon size 3′ boundary^(a)Frame^(c) Primers 1 ND^(d) ND ND AAG|gtgnat ND 2 ND 202 193 AAG|gtaaga 33 tgtcag|T 395 190 CAG|gtaagg 1  3F GAGTTTGCAGTGATGTTGCTATTC (SEQ ID NO:23)  3R ACCGCCTTACCTGCTGCTC (SEQ ID NO: 24) 4 tattag|G 585 70 CAG|gtatat2  4F TGCACTTAATTTTCTTATTACAGTGAG (SEQ ID NO: 25)  4RTAAGGCACACTACTGGGGTG (SEQ ID NO: 26) 5 tgacag|A 655 92 CCT|gtaagt 3  5FGAACACCCCAGTAGTGTGCC (SEQ ID NO: 27)  5R TGAGTCCTAGAGAAGTCACAGACATC (SEQID NO: 28) 6 ttgca|A 747 199 AAA|gtgagt 2  6F GGCTCTTTTCATGGTCTCGTC (SEQID NO: 29)  6R TGTTTTGCTCTCCACCCTTC (SEQ ID NO: 30) 7 ttttag|C 946 122CAG|gtaagc 3  7F GCACTGGATTTTAACAAGGCATG (SEQ ID NO: 31)  7RAGGGCATAGATTGTCCTCGG (SEQ ID NO: 32) 8 ctgcag|C 1068 148 GAG|gtaaac 2 8F TGGGAATACTGATGATGTGCC (SEQ ID NO: 33)  8R CATAACCAGCGAGTCTGCAC (SEQID NO: 34) 9 ccacag|G 1216 132 ATG|gtaacg 3  9F CATTTGGGCATTTCGCATTC(SEQ ID NO: 35)  9R ACCAAACCAAACTCCAGCCC (SEQ ID NO: 36) 10 ttgcag|C1348 156 CAG|gtacta 3 10F TGCCCCCATTGTTCTGCTTG (SEQ ID NO: 37) 10RGGACAGCAGATAAATGGCTCC (SEQ ID NO: 38) 11 ctgtag|G 1504 99 GAG|gtaatg 311F GCATCTCGCATGTCTAATGCCAC (SEQ ID NO: 39) 11R AAGCTGTGATGTCCCCAAATG(SEQ ID NO: 40) 12 tcccag|G 1603 126 CAG|gtgagc 3 12FGACCATGTCCAGTGCAGCTC (SEQ ID NO: 41) 12R CGTTCAGGATCACCACAGCC (SEQ IDNO: 42) 13 tcccag|G 1729 119 AAG|gtacat 3 13F AGTCCTCTGATTGGGGGGAG (SEQID NO: 43) 13R CCATTCTGCACCCAATCAAAAC (SEQ ID NO: 44) 14 tttcag|C 1848403 AAG|gtaatc 2 14F AAAATGGCAGAATGAAAGCACC (SEQ ID NO: 45) 14RCTGATGAACTCCAAAGGTTCTG (SEQ ID NO: 46) 15 ttccag|G 2251 310 AGG|gtaaga 315F GGAAGAGTCAGTGGTGCTCC (SEQ ID NO: 47) 15R CGCCAAAGACCGAAAGGAC (SEQ IDNO: 48) 16 ttctag|G 2561 143 CAG|gtactc 1 16F AGGGTCCTTCTGGCTGCGAG (SEQID NO: 49) 16R GCTGTCAAGCAGCCTCCAC (SEQ ID NO: 50) 17 ttgtag|T 2704 184GAA|gtaagt 3 17F GCTCTCAAGGCAGAAGTGTG (SEQ ID NO: 51) 17RGGAAGGCACCTCTGTAAGTTC (SEQ ID NO: 52) 18 gtccag|T 2888 281 ATT|gtgagt 118F GCTCCTAACCTGTGCCCTTC (SEQ ID NO: 53) 18R GAATTTGACTTCCACAAAGCCC (SEQID NO: 54) 19 ctccag|G 3169 138 TTG|gtatgg 3 19F CGCCCACTGACCACTGTGTG(SEQ ID NO: 55) 19R GAGCCAGAGGAAATGGGTTG (SEQ ID NO: 56) 20 gcacag|G3307 143 CAG|gtaagc 3 20F AGCATTTACCAGGTGAAGTCC (SEQ ID NO: 57) 20RTTGCACACGCCTGCTTAC (SEQ ID NO: 58) 21 tcccag|G 3450 100 GAG|gtcagt 2 21FTGTTCCCGTTTCCTCTTG (SEQ ID NO: 59) 21R GCACAGGAAACACAGCATTC (SEQ ID NO:60) 22 aaatag|G 3550 255 ACT|gtaagt 3 22F GCAGGTAAATGGACAAGAACAC (SEQ IDNO: 61) 22R ACTACCACGGTGGGAAGACC (SEQ ID NO: 62) 23 ctgcag|G 3805 341GAG|gtgagt 3 23F CCCTTCTAACCCACCCTCAC (SEQ ID NO: 63) 23RGACACATCAGCCTTGCTC (SEQ ID NO: 64) 24 ND 4346 ND ND ^(a)Consensussequences for the 5′ and 3′ exonic boundaries are (^(T) _(c))₁₁ncag|Gand AG|gt^(g) _(a)agt, respectively (20). Upper case denotes exonicsequence. ^(b)Exon positions are in reference to the coding sequence ofPTCH (3) with the beginning ATG as nucleotide 1. ^(c)5′exon boundarybegins after the first, second,or the third base of the codon of thetranslation reading frame. ^(d)ND not determined.

One report (Schofield, D. et at., supra) has shown that fivemedulloblastomas (two BCNS-associated cases and three sporadic cases)bearing LOH on chromosome 9q22.3-q31 are all of the desmoplasticsubtype, suggesting LOH on 9q22.3 is histological subtype specific. Wefeel that the conclusion derived from only five positive tumors is a notstrong one because we and others (Raffel, C. et al. (1997) Cancer Res57:842-845) have found nondesmoplastic subtypes of medulloblastomasbearing LOH on chromosome 9q22.3. Independently, another group hasreported their finding of ptc mutations in sporadic medulloblastomas(Raffel, C. et al, supra).

A change of T to C at nucleotide 2990 (in exon 18) was identified in DNAfrom one of nine sporadic meningiomas, causing a predicted change ofcodon 997 from Ile to Thr (Table 2). The meningioma bearing thismutation also has allelic loss on 9q22.3 Blood cell DNA is heterozygousfor this mutation, but DNA from the tumor contains only the mutantsequence. Of 100 normal chromosomes examined, none has this sequencechange, suggesting that this mutation is not likely a commonpolymorphism. This patient is 84 years old and has had no phenotypicabnormalities suggestive of the BCNS, suggesting that this sequencealteration may not have caused complete inactivation of the ptc gene.None of the other eight meningiomas had detectable LOH at chromosome 9q.

TABLE 2 PATCHED gene alterations^(a) Tumor Pathology Nucleotide CodonExon Consequence LOH Mutation Type Med34 Medulloblastoma TC1869A 623 14Frameshift Yes Somatic (desmoplastic) Med36 Medulloblastoma (classic)G2503T 835 15 Glu to STOP Yes Somatic Men1 Meningioma T2999C 997 18 Ileto Thr Yes Germ-line Br349 Breast carcinoma T2863C 955 17 Tyr to His YesSomatic Br321 Breast carcinoma A2975G 995 18 Glu to Gly No Somatic Co320Colon tumor cell line A2000C 667 14 Glu to Ala No Unknown Co8-1 Coloncarcinoma T to C Introl 10 Polymorphism No Germ-line CO15-1 Coloncarcinoma T to C Introl 10 Polymorphism No Germ-like

We also examined a variety of other tumors (10 primary tumors and 1 cellline), 18 bladder tumors (14 primary tumors and 4 cell lines), and 2ovarian cancer cell lines. These tumors are not known to occur in higherthan expected frequency in BCNS patients. We identified sequenceabnormalities in two breast carcinomas and in the one colon cancer cellline (Table 2). The mutation found in breast carcinoma Br349 is notpresent in the patient's normal skin DNA, indicating that the sequencechange is a somatic mutation. Direct sequencing of the PCR productindicated that only the mutant allele is present in the tumor. Thismutation changes codon 955 from Tyr to His, and this Tyr is conserved inhuman, murine, chicken, and fly ptcII homologues (Goodrich, L. V. et al.(1996) Genes Dev 10:301-312). The mutation in breast carcinoma Br321 ispredicted to change codon 995 from Glu to Gly, and the tumor with thismutation retains the wild-type allele. We have sequenced exon 18 in DNAfrom the blood of 50 normal person s and found no changes from thepublished sequence, suggesting that the sequence change found in Br321is not a common polymorphism. Furthermore, examination of the DNA fromthe cultured skin fibroblasts of the patient did not reveal the samemutation, indicating that this is a somatic mutation.

Because DNA is not available from normal cells of the patient from whichcolon cell line 320 was established, we used simplified amplification ofspecific allele analysis (Lei, X. and Hall, B. G., supra) to examine 50normal blood DNA samples for the presence of the sequence alteration andfound none but the DNA from this cell line to have the mutant allele,suggesting that this mutation also is unlikely to be a common sequencepolymorphism. For bladder carcinomas, a newly isolated microsatellitethat was derived from intron 1 of the ptc gene was used to examine LOHin the tumor. Three primary bladder carcinomas showed LOH at thisintragenic locus. With no ptc mutations detected in these tumors, wesuspect that the LOH in these three bladder carcinomas may reflect thehigh incidence of while chromosome 9 loss in bladder cancers (Sidransky,D. et al., supra). A similar observation has been reported previously(Simoneau, A. R. et al. (1996) Cancer Res 56:5039-5043).

We also detected a sequence change in intron 10 in two colon carcinomas,15-1 and 8-1, an alteration that was reported previously as a splicingmutation (Unden, A. B. et al. (1996) Cancer Res 56:4562-4565). Becausewe found the same sequence change in about 20% of normal controlsamples, we suggest that this more likely is a nonpathogenicpolymorphism. The ptc protein is predicted to contain 12 transmembranedomains, two large extracellular loops, and one intracellular loop(Goodrich. L. V. et al., supra). Of the six mutations we identified,four are missense mutations. Three mutations lead to amino acidsubstitutions in the second, extracellular loop, and one mutationresults in an amino acid change in the intracellular domain.

Our data indicate that somatic inactivation of the ptc gene does occurin some sporadic medulloblastomas. In addition, because missensemutations of the ptc gene were detected in breast carcinomas, we suspectthat defects of the ptc function also may be involved in some breastcarcinomas, although biochemical evidence is necessary to show how thesemissense mutations might impair ptc function. Of 11 colon cancers and 18bladder carcinomas examined, we found only one mutation in 1 colon cellline, suggesting that ptc gene mutations are relatively uncommon in donand bladder cancers, although the incidence of chromosome 9 loss inbladder cancers is high (Cairns, P. et al., supra).

Published reports of SSCP analysis of tumor DNA identified mutations inthe ptc gene in only 30% of sporadic BCCs, although chromosome 9q22.3LOH was reported in more than 50% of these tumors (Gallani, M. R. etal., supra). It has been reported that heteroduplex/SSCP analysis ofgene mutations is more sensitive than SSCP analysis (Spritz, R. A. etal., supra). In our studies, we were able to identify a point mutationin the 310-bp-PCR product from exon 15 using heteroduplex analysis,whereas SSCP analysis failed to reveal this sequence change (Table 2).Therefore, we suspect that there may be more mutations in BCCs than wehave found thus far. Analysis of the ptc gene in BCNS patients and insporadic BCCs has identified mutations scattered widely across the gene,and the majority of mutations were predicted to result in truncatedproteins (Hahn, H. et al., supra; Johnson, R. L. et al., supra; Gallani,M. R. et al., supra; Chidambaram, A. et al., supra; Unden, A. B. et al.,supra; Wicking, C. et al. (1997) Am J Hum Genet 60:21-26). In ourscreening, we found two breast carcinomas bearing missense mutations ofthe ptc gene. In one of these two tumors, B349, direct sequencingindicated a deletion of the other copy of the ptc gene. Any comparisonof mutations in skin cancers versus extracutaneous tumors must considerthe wholly different causes of these mutations; UV light is unique tothe skin.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent o application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method of identifying an agent that mimics hedgehog inhibitoryactivity of patched, comprising: (i) providing a culture of mammaliantest cells; (ii) contacting the mammalian test cells with one or moretest agents, wherein the hedgehog/patched signaling pathway is active insaid mammalian test cells, but said cells are not responsive to hedgehoginduction, and wherein said mammalian test cells express a reporter genethat is operatively linked to a transcriptional regulatory sequenceregulated by patched signal transduction, and wherein said mammaliantest cells are not also contacted with exogenously provided hedgehogprotein, and (iii) identifying the one or more test agents that mimichedgehog inhibitory activity of patched relative to that present in theabsence of the identified test agents.
 2. The method of claim 1, whereinthe mammalian test cells are characterized by loss of function of apatched gene.
 3. The method of claim 2, wherein the mammalian test cellscharacterized by loss of function of a patched gene are basal cellcarcinoma cells.
 4. The method of claim 1, wherein the hedgehoginhibitory activity is assayed by examining cell phenotype.
 5. Themethod of claim 1, wherein the hedgehog inhibitory activity is assayedby examining expression of a reporter gene operatively linked to atranscriptional regulatory sequence from patched, Gli, or hedgehog. 6.The method of claim 5, wherein examining expression further comprisesexamining mRNA expression.
 7. The method of claim 5, wherein examiningexpression further comprises examining protein expression.
 8. The methodof claim 2, wherein the hedgehog inhibitory activity is assayed byexamining cell phenotype.
 9. The method of claim 2, wherein the hedgehoginhibitory activity is assayed by examining expression of a reportergene operatively linked to a transcriptional regulatory sequence frompatched, Gli, or hedgehog.
 10. The method of claim 9, wherein examiningexpression further comprises examining mRNA expression.
 11. The methodof claim 9, wherein examining expression further comprises examiningprotein expression.
 12. The method of claim 1, wherein the mammaliantest cells are characterized by loss-of-function of a patched protein.13. The method of claim 12, wherein the hedgehog inhibitory activity isassayed by examining cell phenotype.
 14. The method of claim 12, whereinthe hedgehog inhibitory activity is assayed by examining expression of areporter gene operatively linked to a transcriptional regulatorysequence from patched, Gli, or hedgehog.
 15. The method of claim 14,wherein examining expression further comprises examining mRNAexpression.
 16. The method of claim 14, wherein examining expressionfurther comprises examining protein expression.
 17. A method ofidentifying an agent that mimics hedgehog inhibitory activity ofpatched, comprising: (i) providing a culture of mammalian test cells;(ii) contacting the mammalian test cells with one or more test agents,wherein the hedgehog/patched signaling pathway is active in saidmammalian test cells, but said cells are not responsive to hedgehoginduction, and wherein said mammalian test cells express a reporter genethat is operatively linked to a transcriptional regulatory sequenceregulated by patched signal transduction, and (iii) identifying the oneor more test agents that mimic hedgehog inhibitory activity of patchedrelative to that present in the absence of the identified test agents.18. The method of claim 17, wherein the mammalian test cells arecharacterized by loss of function of a patched gene.
 19. The method ofclaim 18, wherein the mammalian test cells characterized by loss offunction of a patched gene are basal cell carcinoma cells.
 20. Themethod of claim 17, wherein the mammalian test cells are characterizedby loss-of-function of a patched protein.
 21. The method of claim 17,wherein the hedgehog inhibitory activity is assayed by examining cellphenotype.
 22. The method of claim 17, wherein the hedgehog inhibitoryactivity is assayed by examining expression of a reporter geneoperatively linked to a transcriptional regulatory sequence frompatched, Gli, or hedgehog.
 23. The method of claim 22, wherein examiningexpression further comprises examining mRNA expression.
 24. The methodof claim 22, wherein examining expression further comprises examiningprotein expression.
 25. A method of identifying an agent that mimicshedgehog inhibitory activity of patched, comprising: (i) providing aculture of mammalian test cells characterized by loss of function of apatched gene; (ii) contacting the mammalian test cells with one or moretest agents; and (iii) identifying the one or more test agents thatmimic hedgehog inhibitory activity of patched relative to that presentin the absence of the identified test agents.
 26. The method of claim25, wherein the mammalian test cells express a reporter gene that isoperatively linked to a transcriptional regulatory sequence regulated byhedgehog/patched signaling.
 27. The method of claim 25, wherein themammalian test cells characterized by loss of function of a patched geneare basal cell carcinoma cells.
 28. The method of claim 25, wherein themammalian test cells are characterized by loss of function of a patchedprotein.
 29. The method of claim 25, wherein the hedgehog inhibitoryactivity is assayed by examining cell phenotype.
 30. The method of claim26, wherein the hedgehog inhibitory activity is assayed by examiningexpression of a reporter gene operatively linked to a transcriptionalregulatory sequence from patched, Gli, or hedgehog.
 31. The method ofclaim 30, wherein examining expression further comprises examining mRNAexpression.
 32. The method of claim 30, wherein examining expressionfurther comprises examining protein expression.
 33. The method of claim25, wherein the hedgehog inhibitory activity is assayed by examiningexpression of a reporter gene operatively linked to a transcriptionalregulatory sequence from patched, Gli, or hedgehog.
 34. The method ofclaim 33, wherein examining expression further comprises examining mRNAexpression.
 35. The method of claim 33, wherein examining expressionfurther comprises examining protein expression.
 36. The method of claim26, wherein the mammalian test cells characterized by loss of functionof a patched gene are basal cell carcinoma cells.